Depositing fluid specimens on substrates, resulting ordered arrays, techniques for analysis of deposited arrays

ABSTRACT

An apparatus and a method is described for depositing fluid dots on a receiving surface to form an array. The apparatus includes a deposit device cooperatively related with a fluid source, a drop-carrying element coupled to the deposit device, a transport mechanism for positioning the device at a precisely referenced position over the receiving surface, and a drive mechanism for moving the element, relatively, in deposition motion toward and away from the surface.

BACKGROUND OF THE INVENTION

The invention relates to the deposit upon substrates of small quantitiesof fluid in a precise manner and in arrays of desired density andconsistency. The invention is useful, for instance, in carrying outreactions, in providing accurate overlays of deposits, and, inparticular, in preparing microscope slides and membranes with biologicalmaterials.

The invention also relates to array products produced by the noveldeposit techniques and to methods of analysis that employ the deposittechniques.

In the field of biochemistry it is desirable to accurately andefficiently deposit tens, hundreds, thousands and tens of thousands ofsamples of differing compositions on reaction or examination areas.Improvement in the speed of deposition, the precision of the size,shape, quantity and location of the deposits and the control overdensity of the deposits can lead to important advantages.

In particular, well developed biological analytical technology, andrecently developed “Lab on a Chip” or “Gene Chip” techniques requirecreation of dense arrays of fluorescently labeled micro-organisms andDNA assays in a two dimensional field. It is desirable to place thearrays on a conventional microscope slide, and to create many suchslides simultaneously in a manufacturing process.

In important applications, single stranded DNA or PNA or otherbiological elements in the form of fragments carrying known informationare distributed onto the surface of a planar field array containing upto possibly 100,000 objects per microscope slide. The objects of thearray represent discriminating sequence information. Differentlaboratories have targeted the objects of the array to have various spotsizes over a range of the order of 25 to 250 μm in diameter, dependingprimarily upon the total number of objects anticipated in the array. Theobjects of the array are probed with fluorescently labeled fragments ofpotential complementariTy. When a match occurs between these fragmentsand hybridization occurs, a positive is scored by observing fluorescenceat the site of hybridization. By manipulating the deposition ofcomplementary strands or fragments into the array and scoring “hits”,many levels of information can be inferred.

For gene chip technology to proceed to complete fruition, as well as toimprove the application of previous analytical techniques, economicalinstruments have been needed that can rapidly and accurately create thedense array of objects over a large field portion of a glass microscopeslide or slide-like member that occupies an area approximately 22 mmwide and 50 mm long of a slide that is nominally 25 mm×75 mm.

In the deposition upon a microscope slide of 25 discrete, minutequantities of a large variety of fluid materials, the volume depositedat a discrete spot typically may be from a few pico liter to a fractionof a micro liter, depending upon the application. The biologicalmaterial carried in this fluid can range from a few strands of shortoligonucleotides in a water solution to a high concentration of longstrands of complex proteins. The properties of these fluids varyenormously. Some are akin to water while others are far more viscous,resembling a light oil or honey. The range of fluids that may beemployed also exhibits wide differences in evaporative characteristicsand in other properties.

Such large range of property variations in fluids of interest has causedgreat difficulties for any single type of process to operate over a widerange.

Certain processes employing photolithographic techniques have offeredexcellent positional accuracy of the objects and high dot density buthave great limitations due to cost and due to the limited range ofbiological and chemical techniques and substrates that are applicable.These techniques typically construct short segments of DNA or othermolecules by adding single bases, one at a time.

Certain other processes for forming arrays of dots of biologicalmaterial have utilized piezo micro cylinders to aspirate and jet smallvolumes of fluid containing the material while others have usedprocesses akin to a fountain pen, comprising a “quill” deposition tool.An assemblage of quills suck up a desired amount of fluid and by tappinga quill upon the receiving substrate, the meniscus holding the fluid inthe gap of the quill breaks, due to inertia of the fluid within thesuddenly stopped tool, so that a drop of fluid is effectively propelledfrom inside the quill to the impacted surface.

The development of such techniques has occurred against the backgroundof the quite old technique for forming much larger deposits, oftransferring a portion of fluid by a pin or a set of pins that are e.g.dipped in a fixed reservoir containing fluid to be transferred andmoving the pins into position to contact a usually soft substrate toform relatively large spots. Some of these instruments are known as“replicators ”. An example of a product produced by such prior pinswould be a 22 cm×22 cm bioassay plate carrying 0.6 mm diameter spotslocated on a grid 1 mm on center. This spot density is approximately 3orders of magnitude too low from that needed for current “Gene Chip”applications, and the previously known techniques are impractical forpresent purposes for a number of other reasons as well.

SUMMARY OF THE INVENTION

One purpose of the invention is to provide a technology adapted to thedeposition of very small drops of fluids, e.g. drops that form spots ofless than about 375 or 300 μm diameter, and in important cases muchsmaller than that, and at correspondingly high densities (as used inthis application, the term fluid “drop” refers to a very small quantityof fluid, and not to any particular shape of the fluid volume). Thefluids and the resultant dots permissibly exhibit a wide range ofproperties such as viscosity, evaporative characteristics, surfacetension, wettability, surfactant characteristic, dynamic contact angleand free surface energy. These and numerous other objectives areachieved by a number of broad features and preferred embodiments whichare individually novel and important and which in many cases cooperateto achieve highly effective results.

According to one aspect of the invention, an apparatus for depositingfluid dots on a receiving surface in an array suitable e.g., formicroscopic analysis reaction and the like, is provided, comprising adeposit device and a fluid source which are cooperatively related toenable the deposit device to precisely size a drop of fluid of smalldiameter on a drop-carrying surface of the device, transport mechanismfor positioning the device at a precisely referenced lateral positionover the receiving surface and drive mechanism for moving the depositdevice, relatively, in deposition motion toward and away from thesurface, the apparatus adapted, by repeated action, to deposit the dropsof fluid precisely in a desired array, preferably the apparatus beingcomputer controlled.

Preferred embodiments have one or more of the following features.

The drop-carrying surface has a diameter less than 375 micron,preferably less than 300 micron, preferably between about 15 or 50micron and 250 micron.

The drop-carrying surface is bound by a sharp rim that defines theperimeter of the drop of fluid.

The deposit device is a pin or pin-like structure having an end surfacethat carries the fluid drop, preferably the pin or pin-like structurehaving sides that intersect with the end surface to define a sharpperipheral drop-defining rim.

Another broad aspect of the invention is an apparatus for depositingfluid drops on a receiving surface per se, comprising a deposit deviceand a fluid source which are cooperatively related to provide to adrop-carrying surface of the deposit device a precisely sized drop offluid, the deposit device being a pin or pin-like structure having anend surface that serves as the drop-carrying surface, the pin orpin-like structure having sides that intersect with the end surface todefine a sharp peripheral drop-defining rim.

Preferred pins or pin-like structures have an end surface that isgenerally flat and side surfaces that are cylindrical and smooth.

In preferred cases the deposit device is mounted for compliance in thedirection of the deposition motion when the deposit device engages thereceiving surface, preferably the deposit device being compliantlydisplaceable by overcoming resistance of a resilient member or weight,preferably, when the deposit device is an axially slidable pin orpin-like structure, the means for urging comprises a coaxial spring or aweight acting on the pin or pin-like structure. Also preferably, thedrive mechanism for the deposit device is constructed to overtravelbeyond a level at which the compliantly displaceable deposition deviceengages the receiving surface.

In important cases the deposit device, at the time of deposit, islaterally constrained to a reference position, as by the deposit devicebeing mounted on a flexure system that defines the referenced lateralposition of the deposit device, or the deposit device is mounted in amanner permitting its displacement relative to its mounting upon itsengagement with the receiving surface, at the time of engagement of thedevice with the receiving surface, the deposit device being subjected toa lateral force or turning moment that engages the deposit device withat least one lateral reference surface, preferably the deposit devicebeing a pin or pin-like structure which is free to slide axiallyrelative to its mounting upon engagement of the pin or pin-likestructure with the receiving surface, and which is urged against thelateral reference surface by a spring , a weight such as the weight ofthe device, an eccentric weight, or the device being tilted, or byelectrical or magnetic forces acting upon the pin or pin-like structure,to produce a lateral force or moment toward the reference surface.

In many important cases, the fluid source is a mobile fluid storagedevice that is movable relative to an array of deposit locations, thefluid storage device being constructed and arranged to resupply thedeposit device at various locations along the array.

Another broad aspect of the invention is an apparatus for depositingfluid drops on a receiving surface, comprising a deposit device and afluid source which are cooperatively related to provide to the depositdevice a drop of fluid, transport mechanism for positioning the depositdevice over a receiving surface and drive mechanism for moving thedeposit device, relatively, in deposition motion toward and away fromthe receiving surface, the apparatus adapted, by repeated action, todeposit the drops of fluid in a desired array, the fluid source being amobile fluid storage device that is movable relative to the array ofdeposit locations, the fluid storage device being constructed andarranged to resupply the deposit device at various locations along thearray.

In preferred embodiments employing a mobile storage device, the depositdevice and the mobile storage device are constructed to supply drops tothe deposit device in the immediate vicinity of the deposit locationsfor respective drops, preferably the mobile fluid storage device and thedeposit device being coupled for transverse motion relative to the arrayand decoupled for movement of the deposit device toward and away fromthe receiving surface.

In many cases the mobile storage devices are preferably constructed andarranged to be replenished from a remotely located large reservoir.

In many cases a mobile storage device holds a volume of fluid having afree surface into which the deposit device is lowered and raised toobtain a fluid drop, preferably the mobile storage device beingconstructed to store a multiplicity of isolated fluid volumes in thewells of a multiwell plate, the apparatus constructed to obtain itsfluid from a selected volume of the plate.

In other important cases a mobile storage device defines a generallyannular fluid retention surface or ring (the term “annular” or “ring”being used to refer broadly to a member that has opposed or adjacentsurfaces that can hold a mass of fluid between them by surface tensioneffects, accessible to a deposit device), and the deposit device isconstructed to move within the annular retention surface from retractedto extended positions, in the retracted position the drop-carryingsurface of the deposit device being retracted from the surface of fluidretained by the annular surface of the storage device, and in theextended position the drop-carrying surface of the deposit device beingprojected through and beyond the surface of the retained fluid.

Another broad aspect of the invention is an apparatus for depositingfluid drops on a receiving surface in an array suitable for microscopicanalysis, comprising a deposit device and a fluid source which arecooperatively related to provide to a drop-carrying surface of thedeposit device a precisely sized drop, and a drive mechanism for movingthe deposit device, relatively, in deposition motion toward and awayfrom the receiving surface, the storage device defining a generallyannular fluid retention surface, and the deposit device beingconstructed to move within the annular retention surface from retractedto extended positions, in the retracted position the drop-carryingsurface of the deposit device being retracted from the surface of thefluid retained by the annular surface of the storage device, and in theextended position the drop-carrying surface of the deposit device beingprojected through and beyond the surface of the retained fluid.

In preferred embodiments, a member that defines an annular fluidretention surface is associated with a driver that moves the memberrelative to the deposit device to a replenishment volume in which themember is immersed to receive a supply of fluid.

In certain preferred embodiments the deposit device is a pin or pin-likestructure e.g. having one or more of the features described above, thepin or pin-like structure being mounted within the confines of anannular fluid retention surface and arranged to move axially relativethereto.

Preferably, fluid retaining surfaces of the annular storage device havea hydrophilic surface, e.g. a surface roughness of at least 1000microinch or a surface energy greater that about 2500 mN/m, preferablythe surface comprising tungsten, and preferably, e.g. when cooperatingwith the annular member to pick up a supply of fluid the drop-carryingsurface or tip of the deposit device has a surface of surface energygreater than about 2500 mN/m, preferably the surface comprisingtungsten.

The apparatus of any of the aspects and preferred embodiments describedpreferably include a cleaning system and a control system adapted tocontrol relative movement of the deposit device to a depositingrelationship to the receiving surface and a cleaning relationship to thecleaning system.

Another broad aspect of the invention is an apparatus for depositing anarray of dots on a receiving surface, comprising a deposit device in theform of a pin or pin-like structure having an end surface capable ofprecisely defining a small drop of fluid, a source of fluid for thedeposit device, mechanism for moving the deposit device relatively overan array of spaced apart deposit locations of a receiving surface,mechanism for repeatedly moving the deposit device, relatively towardand away from the receiving surface to deposit respective drops of fluidat selected deposit locations, a cleaning system, and a control systemadapted to control relative movement of the deposit device between aresupply relationship to the source, a depositing relationship to thesubstrate and a cleaning relationship to the cleaning system.

In embodiments in which the deposit device is associated with a mobilesupply device that travels with it, the deposit device and mobile supplydevice are preferably movable together to the cleaning system inresponse to the control system, preferably the mobile supply devicebeing an annular member through which the deposit device operates.Preferably the cleaning system has a nozzle for directing a flow of airpast the annular structure, preferably a cleaning or drying stationcomprising a circular nozzle constructed to discharge a conical flow offluid, preferably compressed air, high pressure liquid, an aerosol orheated air against a deposit device or mobile fluid source, preferablythe deposit device being a pin or pin-like structure surrounded by amobile reservoir in the form of an annular member capable of holding asupply of fluid by surface tension effects, the nozzle flows directed todislodge retained fluid, to clean or to dry the respective parts; insome uses preferably an circular storage device is associated with aheater, e.g., an induction heater.

In certain preferred embodiments of the various aspects and featuresdescribed, there are provided a set of at least two of the depositdevices, at least one fluid source for providing a drop of fluid on eachdeposit device, and mechanism for moving the pins together transverselyover an array of spaced apart deposit locations of the receivingsurface, preferably there being at least four of the deposit devicescomprising a deposit head. Preferably the apparatus includes mechanismfor repeatedly moving each deposit device independently, or mechanismfor moving each deposit device simultaneously, relatively, toward andaway from the receiving surface to deposit respective drops atrespective deposit locations on the receiving surface.

For simultaneous actuation, preferably two or more deposit devices aremounted on a common support, driven by a common driver to depositrespective fluid drops on the receiving surface. In cases in which eachdeposit device is associated with a respective storage ring, the storagerings are also mounted on a common support, driven by a common drive;preferably the spacing of the rings corresponds to the spacing of amultiwell storage plate into which the rings are immersed for resupply.In cases in which the deposit device is lowered directly into fluid andraised to obtain its drop, preferably the spacing of the deposit devicescorresponds to the spacing of wells of a predetermined multiwell plate,the multiwell plate being a mobile fluid supply that is constructed toaccompany the deposit device across the substrate. In the case of supplyrings or direct dipping of the deposit devices, preferably in thespacing corresponds to well-to-well spacing of wells of a 96, 384, 864or 1536 well plate, or a spacing of 9 mm or a submultiple of 9 mm.

The various apparatus described preferably have one or more of thefollowing features.

The deposit device and its mounting limits the force of engagement ofthe deposit device upon the receiving surface to less than 1 gram,preferably less than 0.5 gram, preferably to about 0.3 gram.

The deposit device has a natural frequency greater than 10 Hz,preferably greater than 20 Hz.

The motion of the deposit device toward and away from the receivingsurface is damped, preferably by friction or by a damping materialassociated with the support of the deposit device.

The apparatus of any of the foregoing is preferably constructed to mounta number of microscope slides or slide-like structures to serve as thereceiving surface, and a control system is constructed and arranged todeposit drops of fluid in selected locations on the slides or slide-likestructures, preferably the fluid source comprising a source ofbiological fluid.

Another broad aspect of the invention is a fluid deposit assemblymounted on a carrier for depositing minute drops of fluid at selectedlocations upon a receiving surface, comprising a deposit device havingan exposed tip, preferably of diameter of 375 or 300 micron or less,constructed and arranged to carry and deposit drops of fluid upon thesurface, stable lateral reference surfaces or surface portions exposedfor engagement by the deposit device, the surfaces or surface portionsconstructed and arranged to prevent lateral displacement of the depositdevice relative to the carrier when the deposit device is urgedthereagainst and means for urging the deposit device against thereference surfaces or surface portions at least at the time that thedeposit device approaches the receiving surface to deposit a fluid drop,the reference surfaces or surface portions and the means for urgingcooperating to position the deposit tip in a precisely desired positionas it contacts the receiving surface.

Preferably, for depositing fluid drops in a dense array of mutuallyisolated dots, the assembly comprises a fluid source for repeatedlyproviding a discrete drop of fluid on the tip of the deposit device,mechanism for moving the device relatively over an array of spaced apartdeposit locations of a receiving surface, mechanism for repeatedlymoving the device, relatively, toward and away from the receivingsurfaces to deposit respective dots at respective deposit locations onthe surface, preferably the fluid source being a mobile fluid storagedevice separate from the deposit device, which is generally movable overthe array of deposit locations, the fluid storage device beingconstructed and arranged to resupply the deposit device at variouslocations with respect to the array.

In certain preferred embodiments of this aspect also, the deposit deviceis a slidable pin or pin-like structure constructed and arranged to dipinto a volume of fluid carried by a mobile storage device, preferablythe storage device being constructed to store a multiplicity of isolatedfluid volumes, the apparatus constructed to move the supply devicerelative to the deposit device to select the fluid to be deposited,preferably the storage device being a 96 well plate or a plate having amultiple of 96 wells, and also preferably including at least one drivenstage for moving a selected well of a mobile multiwell plate intoregistry with the deposit device under computer control for enablingmotion of the deposit device to dip into and out of the preselected wellto provide a drop of the selected fluid to the device.

In other preferred embodiments the mobile storage device is an annularring as described above.

In embodiments in which the deposit device is a pin or pin-likestructure, it is preferably positioned by engagement with a surface ofrevolution whose axis is disposed at a predetermined relationship to thereceiving surface or substrate, preferably the surface of revolutionbeing in the form of a supporting ledge that supports the device frommoving in its assembly in the direction toward the receiving surface,but from which the device is free to lift in response to contact of thetip of the device with the receiving surface as the supporting ledge anddevice are together moved relatively toward the receiving surface fordepositing a drop, preferably the surface of revolution having a surfaceof form substantially matching the form of the portion of the devicedisposed to engage it, preferably the surfaces being respectivelyconical, each preferably conforming to a portion of the surface of aright cone.

In certain embodiments a means for urging the deposit device againstreference surfaces applies a lateral force or turning moment to thedeposit device, preferably the force or turning moment being applied bya spring bearing eccentrically on the device or by a pushing memberengaged with a remote end of the deposit device, one of the engaged endand pushing member comprising a surface set at an acute angle to an axisof the device, and the other of the surfaces comprising a convexlycurved surface engaged upon the angled surface, preferably the convexlycurved surface defined by a confined ball that bears against theinclined surface, preferably by being pushed by a weight.

In embodiments in which the deposit device is in the form of a pin orpin-like structure, a structure prevents rotation of the deposit deviceabout its own axis, preferably the pin or pin-like structure confined ina complementary space that prevents its rotation about its own axis or adetent prevents rotation of the deposit device, preferably the detentcomprising part of a coil spring which surrounds and is frictionallysecured to the pin or pin-like structure, a protrusion of the springengaging a stop surface that prevents the rotation, preferably thespring also providing axial compliance to the pin or pin-like structure.

Another broad aspect of the invention is a deposit apparatus comprisinga multiplicity of deposit devices as described, mounted for motiontogether in response to a common actuator, preferably the depositdevices comprising deposit pins or pin-like structures.

Another broad aspect of the invention is an apparatus comprising amobile fluid storage device separate from a deposit device and generallymovable over an array of deposit locations, the fluid storage devicebeing constructed and arranged to resupply the deposit device at variouslocations with respect to the array, in one case, preferably the mobilefluid storage device being constructed to store a multiplicity ofisolated fluid volumes, the apparatus constructed to move the mobilestorage device relative to the deposit device to select the fluid to bedeposited, preferably the deposit device being a pin or pin-likestructure constructed and arranged, under computer control, to dip intoa selected volume of fluid carried by the mobile fluid storage device,preferably the mobile fluid storage device being a multiwell platehaving 96 wells or multiples of 96 wells, or a spacing of 9 mm or asubmultiple of 9 mm and preferably the apparatus including a drivenstage for moving the fluid storage device into registry with the depositdevice under computer control for enabling dipping of the deposit deviceinto a preselected fluid volume; in another case preferably the mobilestorage device is an annular ring that retains a supply of fluid bysurface tension.

The invention also features the method of use of all the describedapparatus in depositing fluid drops, especially the fluids mentioned inthe specification.

Another broad aspect of the invention is a method of depositing abiological compound on a substrate or causing biological compounds tointeract with another substance at a predetermined position on asubstrate, including the step of depositing at least one of the compoundor substance in a precisely determined localized spot relative to thesubstrate by mechanically lowering a compliant deposit device,preferably a compliant pin or pin-like structure, to which a drop of thecompound or substance is held by surface tension, toward the substrateuntil the pin or pin-like structure contacts the substrate or apre-applied compound on the substrate and thereafter mechanicallylifting the deposit device away from the substrate under conditions inwhich the fluid drop transfers to the substrate or the pre-appliedcompound on the substrate, preferably the deposit device, whenapproaching the substrate, applying a force to the substrate of lessthan about 1 gram, preferably less than 0.5 grams, preferably about 0.3grams and preferably the drop being less than 300 micron in diameter,preferably less than 200 or 100 micron in some cases preferablysuperposed drops of both a compound and another substance beingsuccessively deposited by the said technique.

Preferably in certain cases the fluid supply of the biological compoundor substance to be deposited by the pin or pin-like structure isobtained by dipping the pin or pin-like structure in fluid, or thedeposit device is supported above the substrate at the deposit locationwithin a ring holding fluid by surface tension, and the pin is movedthrough the ring in the manner that a relatively small drop of the fluidsupply is held by the end of the pin or pin-like structure by surfacetension, preferably in both cases the pin providing a drop-carryingsurface bound by a sharp rim that sizes the drop.

Preferably the fluid to be deposited is fluid selected from a group offluids disposed in a multiwell plate, either a plate which moves acrossthe substrate to be in proximity to the deposit pin or pin-likestructure, or a plate visited by an annular supply ring.

Another broad aspect of the invention is a method of producing arrays offluid dots comprising providing an array of compliant deposit devicesaccording to any of the foregoing claims, the devices preferably beingin the form of pin or pin-like structures, the devices having spacingscorresponding to the well spacing of a 96 well plate, or a plate havinga multiple of 96 wells or a spacing of 9 mm or a submultiple of 9 mm,according to a sampling plan, preferably either dipping mobile annularsupply rings into wells of the plate or dipping the devices directlyinto wells of the plate with which the device is registered to providefluid drops on the devices and transferring the drops to respectivelocations in substantially denser arrays on a receiving surface,preferably the drops being deposited on a microscope slide in a patternof square arrays.

In the various methods, preferably drops of fluid are deposited undercomputer control, by moving at least one compliantly mounted pin orpin-like structure having a drop-supporting surface of diameter lessthan about 375 microns, preferably less than 300 microns, preferablyless than 250 micron, to a selected position and depositing, with thepin or pin-like structure, a desired material.

In the various methods, preferably the receiving surface is fragile, orsoft, preferably the receiving surface is porous or microporous orfibrous, preferably comprising nitrocellulose, nylon cellulose acetateor polyvinylidine fluoride or a gel, preferably the member defining thesoft or fragile receiving surface being mounted on a rigid carriermember, either directly or upon an intermediate soft or resilient buffermember.

Preferably the method is employed to deposit a fluid selected from thegroup of biological fluids described in the specification, preferablythe material being a biological probe or a chemical for reaction withbiological material, a fluorescing material, an ink, dye, stain ormarker, a photoactive material, or a varnish or an encapsulant or anetchant, or a cleaning or neutralizing agent.

Another broad aspect of the invention is the method of depositing abiological fluid with a pin or pin-like structure comprising supportingfluid within a ring by surface tension, and moving the pin or pin-likestructure through the ring in the manner that a relatively small drop ofthe fluid is held by to the end of the pin by surface tension anddeposited on a receiving surface.

Preferably an array of deposits formed by any of the described methodsis microscopically examined with a wide area scanning microscope.

The invention also features an array product comprising deposited dotsof fluid of diameter less than about 375 micron diameter, preferablyless than 300 micron, preferably between 15 or 50 and 250 micron, in adense array in a pattern corresponding to a function of the distributionof wells of a 96 well plate, preferably the dots being spaced from eachother in the array less than three times their diameter, preferably lessthan twice, or about one and one half times the dot diameter thedeposits preferably residing upon a glass microscope slide or on afragile or soft surface, preferably a porous or microporous surface, thesurface preferably comprising nitrocellulose, nylon, cellulose acetate,polyvinylidine fluoride or a gel, the fragile or soft surface preferablymounted on a rigid support directly or via an intermediate soft orresilient buffer member.

BRIEF DESCRIPTION OF THE DRAWINQS

In the Figures:

FIG. 1 is a free body diagram of a deposit pin that is laterallyconstrained by being loaded against a reference surface. FIG. 1 alsoillustrates alternate mobile sub-reservoirs with which the deposit pinis employed.

FIG. 1A is a cross-section taken on line lA-lA of FIG. 1.

FIGS. 1B and 1C are views similar to FIG. 1A illustrating other pinloading arrangements.

FIG. 1D is a diagrammatic perspective view of a deposit pin that has aconical surface mated with a complementary seating surface of a supportplate, while

FIG. 1E is a diagram of a longitudinal segment of the support seatingsurface and FIG. 1F is a diagrammatic view with vectors that analyze thereaction forces of the support seat in response to lateral biasingapplied to the pin.

FIG. 1G is a representation of a spring-mounted and damped deposit pin.

FIGS. 2, 2A, 2B and 3 are diagrammatic side views of combinations ofdeposit pins and cooperating supports that provide lateral constraint ofthe pin, FIG. 2′ is a preferred alternative arrangement of a portion ofFIG. 2, while FIGS. 3A and 3B are cross-sectional views taken onrespective section lines of FIG. 3.

FIG. 4 is a perspective view of a deposit pin mounted by a pair ofparallel spring flexures by which it is laterally constrained andprevented from rotating about its own axis, the pin acting verticallythrough a mobile sub-reservoir while FIGS. 4A and 4B are partialcross-sectional views of other deposit pin mounting constructionsemploying pairs of spring flexures.

FIGS. 5A-5E depict the action of a deposit pin depositing biologicalfluid or reagent, with light contact force, on a precisely locatedposition on a receiving surface such as a microscope slide.

FIGS. 5F-5I depict the action of a pin depositing fluid on a fragile,porous or soft membrane or the like.

FIG. 5J shows a rigid slide carrying a membrane upon which an array ofdots can be deposited.

FIGS. 5K-5N illustrate the process of depositing one deposit uponanother in a precisely aligned manner while FIGS. 5O-5R illustratedeposit of a large spot upon which small fluid dots are deposited.

FIGS. 5S-5V illustrate multiple deposits formed by vertically upwarddeposit motions.

FIG. 6 is a diagrammatic view of depositing dots of fluid onflat-bottomed wells of a multiwell plate.

FIG. 7 depicts a mobile sub-reservoir that travels from one depositposition to another with a separate deposit device illustrated as adeposit pin.

FIG. 8 depicts a system employing the action depicted in FIG. 7,combined with a cleaning station and a central supply of fluid specimen.

FIG. 9 is a side view and FIG. 10 a top view of a deposit head,comprising a deposit pin and an annular sub-reservoir through which thedeposit pin operates, while FIGS. 9A-9D depict a sequence of stages ofthe deposit action of the head of FIG. 9.

FIG. 9E depicts supply or resupply of the sub-reservoir of FIG. 9.

FIG. 9F depicts cleaning the ring and pin of FIG. 9 at a cleaningstation while FIG. 9G depicts a station for removing liquid, washing anddrying the pin and ring.

FIG. 9H depicts the narrow walls of the wells of a PCR plate and thesupply of a sub-reservoir by immersion in a well.

FIG. 9I is a cross-sectional view of a presently preferred annularsub-reservoir device suitable for picking up low viscosity fluids from anarrow main supply as illustrated in FIG. 9H, while FIG. 9J is an endview of the device of FIG. 9I.

FIG. 9K is a magnified view of a pin and ring assembly in which thefluid contact surfaces are specially coated while FIG. 9L illustrates ona less magnified scale the immersion of the assembly for pickup of alocal fluid supply and FIG. 9M, similar to FIG. 9K, illustrates thefluid load that is picked up by the assembly.

FIG. 11 is a perspective view of a multi-pin deposit head mounted forcooperation with a multiwell mobile supply reservoir while FIG. 11Ashows the same elements as FIG. 11 in a supply relationship.

FIG. 12 is a diagram of an operable pin pattern of micro deposit pinswhile FIG. 13 illustrates the initial relationship of the pins to astandard 96 well supply plate.

FIG. 14 defines a useful sampling sequence for the pins of FIG. 12.

FIG. 15 illustrates a pattern of separated squares on a microscope slidewhich the pins of FIG. 12 can simultaneously address while FIG. 15Bshows details of a square;

FIGS. 16, 17, 18, 19 and 19B are views similar, respectively, to FIGS.12-15B, illustrating an arrangement employing a 12 pin pickup array usedwith a 96 well supply plate.

FIG. 20 is a perspective view of an assembled deposit pin constructedaccording to FIG. 2 combined with a respective supply ring; FIG. 20A isa similar view of a group of four such assemblies.

FIG. 21 is a perspective view of a particular device employing detailsof the pin design of FIG. 2, mounted for X,Y and Z travel as anarray-forming device while FIG. 22 illustrates an arrayer system,employing a group of assemblies of FIG. 20A or FIG. 21 constructed forcommercial use.

FIGS. 23 and 23A are perspective views of a combined weak and strongflexure mounting of a deposit pin at respectively different stagesduring operation.

FIG. 23B is a perspective view of the subassembly of FIG. 23 combinedwith drivers for the pin and the sub-reservoir supply ring.

FIG. 24 shows a ganged deposit system having four independently operabledeposit pins.

FIG. 24A is a partial perspective view and FIG. 24B is a plan view of aganged deposit system having a number of flexure-mounted deposit pinsdriven by a single driver and a corresponding number of sub-reservoirrings driven by a single driver; FIG. 24C is a plan view of an array ofdots producible by the system of FIGS. 24A and 24B.

FIG. 24D is a perspective view of the ganged system driven by a linearstage.

FIG. 25 is a perspective view of a machine for depositing dots ofbiological fluid in dense array upon a series of microscope slides.

FIG. 25A is a side view of a slide holding arrangement useful in themachine of FIG. 25.

FIGS. 26 and 27 show features of the control system software and methodfor conducting the deposit action.

PREFERRED EMBODIMENTS

In preferred embodiments a deposit pin of small cross-section isemployed with a mobile fluid reservoir to which the pin is repeatedlyexposed, the pin being sized and shaped to define and retain on its tipa drop of fluid from the reservoir, the drop containing only enoughmaterial to deposit a single dot.

The volume of the drop is typically determined by pin cross-sectiondimensions, shape and surface characteristics of the tip of the pin aswell as by the viscosity and surface tension of the fluid to bedeposited and the techniques by which the tip is supplied.

By providing the tip of the deposit pin with a sharply defined rim, itis found during repeated action that fluid drops of consistent volumeare defined by the tip when all other factors remain constant.

Presently we prefer that the rim of the tip of the pin be “square”, i.e.that, in profile, the end surface of the tip of the pin be substantiallyat a right angle to the side surface of the pin, and that the pin sidesurfaces be smooth. Preferably the pin is round in transversecross-section, though it may be of other shapes. Under certaincircumstances, as when depositing on porous substrates that readilyreceive the fluid, the end surface of the pin may also have asurface-tension enhancing surface to optimize the fluid acquisitioncapability of the pin. For example it may have a roughened surface,surface roughness of at least 1000 microinch, or be composed of highsurface energy material, (surface energy greater than 2500 mN/m), suchas tungsten.

It is found that arrays of fluid dots between about 20 microns to 375microns can be deposited using biologic fluids of conventionalconcentrations, by employing deposit pins that have, in their tipregions, a wire or wire-like geometry of diameter (true diameter orcross-section dimension) between about 0.001 inch (25 microns) and 0.015inch (375 microns). The smaller tips, i.e. tips smaller than 0.012 inch(300 microns) are referred to here as “microtips”. A preferred range oftip sizes is between 50 microns and 250 microns.

In preferred embodiments, at least at the time of engagement of the pinagainst the receiving surface, precise positioning of the pin isachieved by lateral constraint of the pin to a reference position. Thepin is also axially controlled, preferably by a compliant mounting, tolimit the maximum deposit force to preferably less than 1 gram, inpreferred cases less than 0.5 gram, e.g. 0.3 grams. The lateralconstraint ensures that the deposited drop is precisely located whilethe “soft landing” assists in achieving a well-defined deposit, inprotecting the end geometry of the pin to preserve its drop-sizingfunction over long usage, and in protecting fragile substrates that maydefine the receiving surface.

With these provisions, tightly packed arrays of deposited dots of fluidcan be achieved, i.e. arrays with center-to-center spacing between dotsof less than three times the dot diameter, often only twice or one andone half times the dot diameter.

FIG. 1 is a free body diagram of a deposit pin 12 having a microtip thatis laterally constrained to a reference position by application offorces (see FIG. 1A). This achieves precise X,Y positioning of the tipat the time of its engagement with the receiving substrate 20. Tip 12 dhas sharp rim 12 f, and is round in cross-section, of diameter d in therange greater than about 15 microns and less than about 375 microns,preferably less than 300 microns, preferably in the range between about15 or 50 microns and 250 microns. It is capable of defining anddepositing upon substrates micro dots of fluid of generallycorresponding dimension in the tightly packed arrays.

In the embodiment of FIGS. 1 and 1A the deposit pin is carried onsupport 17. The pin, though laterally constrained at the time ofengagement with the substrate, is mounted to be axially compliant, freeto be displaced upwardly relative to the support when the pin encountersthe substrate. When the support is lifted from the substrate the pin isfree to return to its seat. The pressure applied to the substrate by thepin can be determined by the weight of the pin alone, or as supplementedby a spring or added weight. Preferably the pins are secured againstrotation to ensure repeatability of position despite possible variationsin shape of the pin due to manufacturing tolerances.

The details of preferred laterally referenced pins axially displaceablefrom their support are described later with reference also to FIGS. 1Bto 1F and to FIGS. 2-2B, FIG. 2′ and FIGS. 3-3B. With the mountingsshown, the movements occur accurately over a wide range of ambientconditions.

Another laterally referenced pin mounting arrangement is shown in FIG.4, see also FIGS. 4A and 4B. Pin 12 is supported by a pair of spacedapart, parallel, planar, cantilevered flexures 70, 72 that extendperpendicular to the direction of the desired compliant motion of thepin, to provide a parallelogram type of mounting that laterallyconstrains the pin to a reference position to enable landing of the pinat a precise location on the substrate. A soft landing occurs due tocompliance provided by the flexures. To deposit precise arrays of dotsas small as 25 micron (0.001 inch) diameter, an element of spring metalin at least one of the flexures 70, 72 ensures that the deposit pinreturns to its original vertical position after each deposit.

Provision of a suitable mounting and drive of the deposit pin, asillustrated in the FIGS. 1-4, enables a low and predictable contactforce upon the receiving substrate (the “soft landing”) despitevariations in the height of the substrate, e.g. due to variations inthickness of microscope slides or slide-like members upon which thefluid dots are applied. Superior results can be obtained by controllingthe deposit pin force upon the substrate to less than the order of onegram, or 0.5 gram, preferably about 0.3 gram.

While vertical reciprocation of a deposit pin is presently preferred,other motions can be employed.

The deposition systems described have long-term dimensional stability,being immune from temperature, humidity and other ambient changes. Thesystems enable spotting of, e.g., a full set of 40 microscope slideswith 10,000 spots per slide, a process that may require a few hours to afew weeks, depending upon the number of pins operating simultaneously inone head. The instrument may operate unattended for many hours at atime.

For high speed operation, the system also preferably has a naturalresonant frequency higher than 10 Hz, e.g., 20 Hz, which is achieved byemploying a pin of low mass and a suitable support.

To operate at high speed, e.g. such as a drop formation/drop depositcycle of 0.1 second, in addition to having a natural frequency higherthan 10 Hz or 20 Hz, the pin mounting system has a provision for dampingthe motion of the moving pin element, preferably by an amount close tocritical damping. This damping prevents the pin from bouncing anddegrading the spotting process and enables the pin to be moved quicklyaway after each deposit action. In preferred embodiments, damping isobtained concurrently with providing very high compliance of the pinsupport. The general principle of combined compliance and damping isillustrated in FIG. 1G. The actuator A acts through a highly compliantsupport spring Z, buffered by a damping device X, the moving assemblyhaving a natural frequency in excess of 10 Hz. Where sliding action ofweighted pins is employed, friction of the sliding surfaces can beemployed to provide damping.

In embodiments that employ cylindrical deposit pins moving axiallynormal to the deposit surface, with spring mounting systems in which theweight of the pin is insignificant or counter balanced, it is observedthat a spring support system for the pin with stiffness of less than 5gram per millimeter deflection, measured at the pin, produces goodresults for cases in which the amount of pin deflection is a few tenthsof one millimeter. In one particular case, a spring system having aspring deflection ratio of 3 gram/mm, deflected about 0.2 mm, resultedin deposition of dots of fluid of excellent, repeatable quality over arange of microscope slides.

Use of the deposit pins to deposit biological fluid or reagent on arigid substrate is illustrated in the sequence of highly magnified,diagrammatic FIGS. 5A-5E while FIGS. 5F-5I illustrate use of the pins todeposit fluid dots upon a delicate, soft or porous membrane and thelike.

In FIG. 5A, the deposit pin P is seen supporting a drop F of fluidobtained e.g. from a mobile sub-reservoir MW (FIG. 1) or sub-reservoirring 14, (FIG. 1, 4). The pin moves under control of driver D toward aselected target point S on receiving surface R. In the case of dippinginto sub-reservoir MW, surface tension effects hold fluid drop F insubstantially semi-spherical form on the sharp-rimmed tip of the pin,see FIG. 7. When the tip is plunged through fluid held in sub-reservoirring 14, the drop F is normally shallower, less rounded, see FIG. 9.Surface R . when a microscope slide, is typically impermeable andnon-wettable such as silene-coated glass.

In FIG. 5B, the pin has advanced sufficiently toward the receivingsurface R that contact of the fluid drop with the surface has occurred.The drop has been forced to distort to a generally cylindrical shape, C.

In FIG. 5C, the pin P has advanced toward surface R to level L. Thefluid cylinder C₁ is of expanded form, in which its boundary has beenstretched, but it remains as a coherent fluid mass between and in theimmediate vicinity of the receiving surface and the tip of pin P. Thesystem for driving the pin limits the further movement of the pin towardthe receiving surface R in the manner that the maximum force exerted bythe pin upon the substrate is limited, as described.

In preferred mechanical systems, the pin is compliantly mounted andresponds to resistance force transmitted to the pin by the fluid ormechanical contact with the substrate, so that the tip of the pin stopsdespite overtravel of the driver.

In other systems, based upon position detection, the driver is stoppedin response to a position sensor at the desired level. In hybridsystems, combinations of compliant or mechanical limiting systems andpositional detection can be employed.

In FIG. 5C the fluid cylinder C₁, is shown expanded relative to its basein FIG. 5B. The degree of expansion and the curvature of the fluid wallis determined by the degree of wettability of surface R and the surfacetension characteristics of the fluid, as well as by the force applied bythe pin.

In FIG. 5D, the pin P has moved away from surface R, leaving drop DF attarget point S. The drop DF may contract in base diameter. The degree ofsuch contraction or of expansion is determined by the wettability of thereceiving surface and surface tension characteristics of the fluid. Whensurface R is hydrophobic, the deposited fluid drop may contract as itdries, while with wettable, fibrous, or porous surfaces, it may expand;in either event, the size of the deposited dot is determined principallyby the size of the tip of pin 12.

In FIG. SE the pin P, substantially devoid of fluid, moves away fromreceiving surface R, with a component of lateral movement, M. It israpidly resupplied and proceeds to the next target point. The pin P isresupplied in important cases from a mobile local sub-reservoir thataccompanies the pin across the substrate, e.g. the movable reservoir MWof FIG. 1 or the annular supply ring 14 of FIG. 1 or FIG. 4. The depositcycle is then repeated at another lateral (X,Y) position to which thepin is moved.

Instead of placing deposits on a rigid, smooth substrate, the substratemay be a porous or microporous membrane or a delicate film such asnitrocellulose, cellulose acetate, polyvinylidine fluoride (PVDF) ornylon, or it may be an agar gel or other gel. The particular substratecan be selected in accordance with the nucleic acid, protein or othertransfer procedure being employed, and can be the same as or take intoconsideration the substrates previously used in development ofhistorical reference data with which the results of the presentexperiment are to be compared. The compliance of the pin protects suchfragile substrates from damage.

Action of a deposit pin in depositing biological fluid or reagent on adelicate, soft or porous substrate is illustrated in highly magnifiedFIGS. 5F-5I. A delicate, relatively soft membrane D_(M) is supporteddirectly on a rigid support R_(S).

In FIG. 5F, as in FIG. 5A, the tip of deposit pin P having a sharp rim12 f supports a precisely sized drop F of fluid specimen or reagentobtained from a mobile sub-reservoir, and moves toward a preselecteddeposition point S.

In FIG. 5G, the pin has advanced sufficiently toward the receivingsurface R that the fluid drop contacts the surface. The slight pressureof pin P slightly deforms membrane S_(M) as illustrated in FIG. 5G, butaxial compliance of the pin prevents damage to the relatively softsubstrate. Depending upon the porosity or softness of the membrane andany capillarity, the fluid may migrate slightly to the sides, but thesize of the deposited dot of fluid is principally determined by the sizeof the pin's tip.

In other cases, a protective buffer member, not shown, which may be softor resilient may be interposed between deposit pin and substrate. Insome cases where the buffer member is significantly compliant,compliance of the deposit pin itself may be omitted in those conditionswhere a degree of compliance is required.

In FIG. 5H the pin P, substantially devoid of fluid, moves away from thereceiving surface with a component M of lateral movement, and proceedsto the next target point. Because of the light contact pressure, theshape of the tip and the presence of the intervening fluid, no particlesof the substrate remain on the tip. Upon relief of the slight depositpressure, the membrane begins to recover its original conformation.

In a short while, as shown in FIG. 5I, the delicate membranesubstantially recovers its conformation and the fluid has dried, leavingdried deposit D_(D) of substantially the size of the tip of the depositpin.

FIG. 5J depicts a glass microscope slide or slide-like member M_(S)carrying a delicate membrane, D_(M), e.g. a thin layer of microporousnitrocellulose film formed by casting, such as is available from GraceBio-Labs Inc. under the trademark Oncyte® Film Slides. The process justdescribed may be employed to deposit microarrays of fluids on suchmicroporous film. Although the deposit-receiving surface is not rigiditself, by virtue of a rigid backing, the delicate membrane can beautomatically processed, scanned, etc. by available robotic equipmentthat engage the rigid frame.

High density arrays of individual deposited dots as depicted in FIGS.5A-5D and 5F-5I are achieved by repeated deposit action of one or moredeposit pins, with selected fluids being deposited at selected preciselocations under computer control as further described below.

FIGS. 5K-5N depict diagrammatically, in magnified scale, the depositingof one deposit upon another in a precisely aligned manner, made possibleby the positional accuracy of the systems being described. FIG. 5K showsa deposit 100, as produced by techniques previously described employinga deposit pin, or by some other technique that achieves a knownposition. FIG. 5L shows deposit pin 12 having been indexed into precisealignment with dot 100, and lowered to engage fluid drop C₁ with it.

FIG. 5M shows the deposited second drop 104 still in fluid state whileFIG. 5N shows dried second dot 106 deposited upon dot 100.

In similar fashion FIGS. 5O-5R illustrate deposit of a relatively largespot of fluid using large deposit pin 120 and subsequent deposit ofsmall drops using a smaller deposit pin 12 having a microtip. The largedrop on the pin 120, in FIG. 5O, forms a large deposited drop 110, FIG.5P, which dries to form a large dried spot 112, FIG. 5Q. Subsequently,small drops 114 are deposited in selected precise locations upon thelarge spot 112, FIG. 5R, which can be identified to computer memory andemployed at the time of microscopic examination to correlate theresults.

FIGS. 5S-5V illustrate the possibility, with selected receivingsubstrates and fluids, of conducting the operation of FIGS. 5K-5N ininverted fashion. Similarly the other deposit actions described may alsobe performed with inverted motions, or at other angles.

Referring to FIG. 6, a deposit device comprising deposit pin 12 andsub-reservoir ring 14 supported by rod 15, is used to deposit dots offluid on the flat bottom F_(B) of a conventional well of a microtitreplate M_(p). A number of precisely located deposits of fluid D_(f) canbe made, taking advantage of the long length and small diameter of thedeposit pin 12, which enables reaching the bottom of the well atprecisely spaced locations. For instance, a series of probes may bebound as dots to the bottom of a well at known, recorded positions, afluid containing the analyte may be used to fill a well, and subsequentto a reaction or incubation interval, the bottom of the well may bescanned by a microscope, or otherwise examined, to determine whichdeposited probes matched the analyte fluid. By pre-preparation of such aplate with known sets of probes, many fluids or many probe actions witha given fluid may be assessed.

Details of preferred embodiments that laterally constrain the pin to areference position and facilitate highly accurate X,Y positioning of amicrotip during each deposit will now be described.

Referring to FIG. 1, deposit pin 12 comprises a relatively large body 12a and a lower portion 12 b of reduced dimension that leads to deposittip 12 d having sharp rim 12 f. The pin also has an upwardly extendingguide portion 12 c.

Large body 12 a provides a downwardly-directed annular surface 12 e thatis engaged upon a support, to receive support force F_(s) that bears thepin's weight.

The assembly is constructed to enable a lateral bias force F_(b) to urgepin 12 against a pair of reference surfaces Ref₁ and Ref₂ which lie atan angle to each other as viewed in horizontal cross-section, FIG. 1A.These reference surfaces are arranged to resist movement of the pin in Xand Y coordinates by applying reaction forces that have X and Ycomponents. The combined X components of the forces provide resistanceforce Fr that balances bias force F_(b). The reference surfaces areconstructed to leave the pin free to move axially along axis A (Zdirection), as by sliding, to provide axial compliance to the tip 12 dwhen the substrate is encountered.

In certain practical embodiments, for mounting the pins, two verticallyspaced horizontal plates 9 and 11, shown in dashed lines, are joined toform carrier 17 that moves in X,Y and Z directions for carrying the pinthrough deposit, cleaning and resupply motions. The upper plate 9 is ata selected distance from the lower plate and applies a constrainingforce F_(c) to constrain the angle of the pin, and hence the position ofits tip 12 d, within selected tolerance.

Lowering carrier 17 causes the precisely positioned tip 12 d to engagesubstrate R, whether the substrate be found at level 20, 20 a or 20 b inthe design range. Upon engagement with the substrate, the pin stops.Further downward movement of the carrier 17 occurs with the compliantpin remaining stationary above its seat, while reverse movement of thecarrier causes the pin to reseat on its support. In this way thevertical movement of the carrier need not be controlled with highaccuracy, and proper contact with the substrate can occur over a rangeof tolerances in the height of the substrates.

The lateral bias force F_(b), for laterally constraining the pin to areference position, can be applied to deposit pin 12 in numerous waysthat permit axial movement of the pin relative to its carrier 17 whenthe pin encounters the substrate. FIG. 1B illustrates application oflateral bias force by miniature spring-loaded bearings that urge the pintoward an inside corner defined by reference surfaces Ref₁ and Ref₂, butleave it free to move axially. FIG. 1C illustrates tilting thelongitudinal axis A of the pin relative to the vertical, in a mannerthat the weight of the pin applies to itself a slight turning momentthat biases the pin against reference surfaces Ref₁ and Ref₂ but leavesit free axially.

Other techniques include introducing bias by an eccentric load or byother loading techniques. Magnetic attraction can also be employed todraw the pin to a defined corner or a particular arc of a conical seat,using permanent magnets or electromagnets. Likewise, electrostaticforces can be employed, e.g. by imparting a charge to a region of one ofthe members relative to the other and employing a dielectric layer toprevent discharge so that the attraction persists at the time ofapproach of the deposit pin toward the substrate. The vertical effect ofsuch loading techniques can be sufficiently small to be overcome byaxial force on the pin, to enable axially compliant motion of the pin.

FIGS. 1D, 1E and 1F show that tangent planes P₁ and P₂ to a segment of aconical seat (FIG. 1E) against which a pin is urged effectively definetwo reference surfaces set at an angle to one another, that act in themanner as explained to resist lateral movement of pin 12 in both X and Ydirections. The same is true for other surfaces of revolution thatdefine seating surfaces. Preferential seating upon a given segment ofthe seating surfaces may be achieved by the loading techniquespreviously described.

FIGS. 2, 2A and 2B, show embodiments that employ a surface of revolutionfor supporting the pin in a laterally constrained manner.

Fluid deposit pin 12, (associated with a fluid supply such as mobilemultiwell plate MW or associated supply ring 14), is constrained betweenupper and lower plates 9 and 11 of carrier 17. Carrier 17 moves in thedirection of arrow Z for supply and deposit actions. In each case ofFIGS. 2, 2A and 2B, an enlarged portion of the pin 12 a rests normallyin a seat 13, 13 a or 13 b in plate 11 which bears the weight of thepin. The pin is free to be displaced relatively upwardly from its seat,as shown, upon contact of tip 12 d with substrate 20.

When the pin 12 rests in its seat, the X,Y position of tip 12 d of thepin is defined by the degree of perfection of the pin 12, the relativedistance to the upper supporting plate 9, the clearance between upperportion 12 c of the guide pin and the guide hole 19 in plate 9, and, asindicated above, a preferred feature in the system that applies adefinite (though permissibly slight) lateral bias of the pin to a givenside of the engaging structure. For this purpose, in FIG. 2, compressionspring 22 is disposed between upper plate 9 and upwardly directed ledge24 of pin 12. The spring is fixed in position and applies its downwardforce with slight and predictable asymmetry relative to center axis A,to bias the pin to a given side, to ensure repeatable positioning of thepin on the same region of its seat on plate 11. Spring 22 is sized andarranged to also provide compliant pressure of tip 12 d of the pin onthe substrate 20, taking into consideration also the mass of the pin.

In the arrangement of FIG. 2′, the lower part of spring 24′ tightlyengages about the pin to secure it rotationally, while an upper leg 22 aof the spring extends into a hole in plate 19, to serve as a stop. Inthis way the rotational position of the pin is secured so that variationin its shape will not introduce variations in position during repeatedactions.

The sizing and nature of the spring can be selected to provide a highnatural frequency for the system. In the various embodiments thus fardescribed, the pins are axially slidable relative to their supports andfriction contact produces a desirable degree of damping of the motionduring rapid reciprocation. In other cases, damping material may beassociated with the spring or other mounting of the device.

In the arrangements of FIGS. 2A and 2B the pin is also biased laterally,e.g. by use of spring 22 or 22′, by angling the long axis of the pin afew degrees from vertical (with the axes of all adjacent pins beingparallel when an array of pins is employed), etc.

The pins of FIGS. 2, 2A or 2B in cylindrical form are readily formableto a suitable degree of perfection. The distance D (FIG. 2) is readilyselectable, considering that, for a given clearance allowance betweenthe hole of the upper plate and upper pin portion 12 c, increase ofdistance D decreases the possible disturbance of the pin from itsnominal orientation due to manufacturing variation, etc. Thus, while thebottom plate 11 of these embodiments defines the vertical level of thetip of the pin 12 d, the top plate 9 is located sufficiently above plate11 that its spacing and the angles produced establish the lateralposition of the tip of the pin within desired tolerances for precisedeposit of dense arrays on the substrate 20.

It is seen from FIGS. 2, 2A and 2B that the driven pin carrier structure17 travels downwardly sufficiently to ensure that tip 12 d can reach thelowest level 20 b of the range of permissible levels. As in previousembodiments, the tip 12 d is axially compliant in the sense that the pincan yield in position so that, when encountering the substrate, itexerts only a controlled light pressure on the substrate before it liftsfrom its seat.

In FIG. 3, the enlarged part 12 a′ of pin 121 and seat 13 c havecomplementary conical surfaces normally engaged unless the tip 12 d′ isengaged with the substrate (but shown disengaged for purposes ofillustration). The upper end surface 12 g of the enlarged body portion12 a′ is sloped in a selected direction as explained further below and arigid ball bearing 38 bears on the sloped surface at a point offset fromcentral axis A. A weight 39 rests upon ball 38, being housed by a borein spacer block 41 upon which the upper and lower plates 9 and 11 areaffixed. The spacer block is advantageously of a low-frictionengineering plastic such as Delryn. The weight 39 is of selected size toadjust compliance to the degree desired for the deposit tip 12 d′ and toapply a slight turning moment M_(o) to the pin toward a portion of theconical seat, (see FIG. 1), via the eccentrically located ball.

As seen in the cross-section of FIG. 3A, weight 39 is of cylindricalconfiguration, free to rotate about axis A with turning of the ball toavoid applying undue drag. The main body 12 a′ of the pin, however, isof square cross-section and is disposed in square channel 42 in spacerblock 41 of like configuration. This prevents pin rotation so thatorientation of the upper end face 12 g and top 12 d′ remain constant.The slope of surface 12 g relative to axis A and the flats of the squaresection of the pin are cooperatively related to cause engagement withsquare channel 42. This is accomplished by sloping surface 12 g toward acorner of the square channel. Thus deposit tip 12 d′ resides at aconstant, precisely defined lateral position by cooperation of eccentricweight 39, the segment of the conical seat against which the pin isurged by the weight, and by the prevention of pin rotation. By use of aselected weight (instead of a spring), the spotting force upon thesubstrate is constant over the range of possible heights of thereceiving substrate, which can enhance repeatability of spot size overthat range.

Such lateral arrangements are important when employing microtips inarrays that require precise positioning such as high density arrays.

The features previously discussed, i.e. lateral reference of the pin,axial compliance, stability, high natural frequency, and damping canalso be simultaneously achieved by flexure mounting of the deposit pinas shown in FIG. 4A. Two similar and highly compliant planar flexures 70a, 72 a have similar elasticity, but one of them, flexure 70 a, is madeof a highly stable material, e.g. metal spring, while the other flexure,72 a, provides good damping properties.

The stable flexure 70 a is preferably manufactured by photo etching athin metal sheet, such as 0.002-inch thick stainless steel, whichexhibits high stability and low rigidity but has poor dampingproperties. The other flexure 72 a, preferably equally compliant, isprovided with desired damping properties, and is less stable. The secondflexure 72 a, for instance, is constructed as a bonded sandwich of twoidentical photo-etched thin plastic sheets 61 such as 0.005 inch thickpolyamide resin, e.g. Kapton® from dupont. An energy absorbent bondingagent, e.g., of thickness t of 0.002″ provides a damping layer 77between these resin sheets. The bonding agent may be a thin coat ofrubber cement such as available from 3M as ID # 62-60065-4826-1 or 3Mdouble sided tape # 927.

In an alternate construction shown in FIG. 4B, flexures 70 b, 72 b areidentical, each being a sandwich of one metal layer 73 and one resinlayer 75 bonded together by rubber damping layer 77. Compliance similarto that of FIG. 4A is achievable with the selection of material ofappropriate thickness, such as either a stainless steel layer 0.0016inch thick or a copper-beryllium layer 0.0022 inch thick, bonded by thedamping layer to a polyamide layer 0.005 inch thick.

The physical properties of the flexures can also be tailored to theparticular need by change in geometry of the flexures. An example is theprovision of cutouts.

In manufacture, a large-area bonded sandwich of all three materials maybe fabricated and the shape of the flexures can then be produced byphoto etching the desired outline and any cutouts.

In a cluster of deposit pin assemblies of the type shown in FIGS. 4, 4Aand 4B, such as shown in FIGS. 24A and B described below ; with 9 mmspacing of pins to correspond with the spacing of wells in a 96 wellplate, the flexure elements are preferably 8 mm wide and 22 mm inlength, and two or more of the pin and flexure assemblies are mounted inparallel, side by side, at 9 mm pin spacings. Preferably two sets ofsuch assemblies, disposed head-to-head as described below, are employedat 9 mm pin-to-pin spacings, so that an X,Y array of pins is achieved.

B. Mobile Fluid Reservoirs and Interaction with Deposit Pins

For making a succession of deposits of the same fluid, as when preparinga number of microscope slides or membranes or providing redundantdeposits on a single substrate, a mobile sub-reservoir, periodicallyre-supplied from a stationary central supply, travels with a depositdevice to be near the deposit locations.

As illustrated in FIG. 7, a deposit head comprises the deposit pin P ofFIGS. 1 or 4, and the sub-reservoir SR which is sized to containsufficient sample to enable deposit of a number of dots before beingresupplied.

After deposit of drop F at target S, e.g. on a microscope plate R or aplate carrying a delicate or soft membrane, the assembly proceeds toplate R₁, pin P is resupplied with drop F₁ by being dipped into andraised from the accompanying sub-reservoir SR, the new drop is thendeposited at target point S₁ at plate R₁, and so on.

The system is especially useful for preparing a number of microscopeslides or membranes as illustrated in FIG. 8. The central fluid supplyCS advantageously is a multiple well plate as conventionally used inmicrobiology, such as a 96 well plate. Cleaning and drying stations CLare also provided. The deposit sequence includes moving the assembly ofdeposit device and mobile sub-reservoir under computer control throughcleaning and drying stations CL, thence to central supply CS at whichthe sub reservoir SR is supplied with a selected fluid sample, e.g. froma selected well of a 96 well plate. Then the group moves over a seriesof receiving surfaces R-R_(n), for deposit of fluid dots at selectedlocations on each, also under computer control. This sequence isrepeated a number of times, with controlled selection of different fluidsamples (from, e.g., different wells of the central supply CS) forrespectively different locations on the plates R or other receivingsurfaces. Data that correlates locations with respective specimens isrecorded in memory and used in subsequent scanning or reading.

The technique of using a deposit tool that accurately sizes eachindividual drop, such as the deposit pin with square rim profile at itsmicrotip, combined with a mobile local sub-reservoir that accompaniesthe tool and carries a volume sufficient to supply a sequence ofdeposits, has a number of important advantages. The technique, based onsmall motions, saves time in avoiding repeated travel to a centralsupply; it avoids evaporation losses of long travel, so that the dropcreated can be very small and the deposited array very dense; and thedots can be kept consistent in size and concentration or biologicalcontent across the array of dots being deposited. The time overheadinvolved in cleaning, transporting and picking up the specimen is keptsmall so that, overall, deposits can be made very fast, inexpensivelyand of desired small size.

In this way a large number (for instance ten to one hundred) identicalmicroscope slides or membranes can readily be prepared by repeatedmotions over an array of the slides or membranes. Each substrate cancarry dots of many different fluids based upon resupply of thesub-reservoir from different wells of a number of multiple well platesintroduced to the system.

The sub-reservoir and the deposition device are decoupled, in beingmovable relatively to one another for resupply and for deposit, as wellas being coupled or at least coordinated, to move laterally over thereceiving surface to produce the series of deposits. The sub-reservoircan move into a resupply position, e.g. by immersion into a well, orunder a suitable pipette. It can be made to hold sufficient fluid inexcess of that required for the sequence of deposits, or to expose asufficiently limited evaporative area, that concentration of thesubstance of interest in the fluid is not substantially affected byevaporation during the multiple deposit sequence.

Thus we have described deposit devices constructed to precisely define asingle fluid drop of desired size, obtained from a mobile sub-reservoir,deposit the drop at a precisely positioned, discrete location and returnby local movement to the sub-reservoir for another drop. In thepreferred embodiment of FIGS. 7 and 8 an axially reciprocable depositpin, as illustrated in FIG. 1, is employed for this purpose inconjunction with an accompanying sub-reservoir in which the pin isdirectly dipped. Alternatively, a probe that dips into a localsub-reservoir as by coordinated rotational or translational motions of awire or pin, can accomplish this action, as can other designs.

Referring now to FIG. 9 and 10, another preferred mobile sub-reservoiris an annular ring 14 which, as depicted, holds fluid between itsinterior opposed surfaces by surface tension effects.

Deposit pin 12, having a sharp rim 12 f at its tip, of diameter dselected to produce the desired size of the deposited dot, is mounted inaxi-symmetric relation to sub-reservoir ring 14. Ring 14 has an internaldiameter d₁ significantly larger than the pin diameter such that fluidspace fs exists between the pin and the inner surfaces of the ring. Asshown in FIGS. 9E and 10, the outer diameter d₂ of ring 14 is sizedsmaller than the well 19 of a central supply plate, 21, so that the ringcan be immersed in it for supply.

During the deposit sequence of FIGS. 9A-9D the ring 14 is heldstationary by its support rod 15 while the deposit pin 12 is moved by anassociated driver D through a sequence of vertical positions. In thestart position, the end 12 d of pin 12 is drawn above the lower surfaceof the retained fluid R_(f) held by surface tension effects between theinternal surfaces of ring 14. This is shown in FIG. 9A. (The pin, forillustration, is shown withdrawn fully above the retained fluid R_(f),although that is not necessary.)

Comparing FIG. 9A with FIG. 9, by downward movement of the pin tip fromabove the lower surface of the retained fluid R_(f) (FIG. 9A), to belowthat surface (FIG. 9), the tip of the pin, with its sharply defined rim,picks up from the retained fluid R_(F) a precisely sized volume of fluidas drop F. The drop is then deposited in the sequence shown in FIGS. 9Cand 9D.

At the resupply position of FIG. 9E, the annular ring 14 is moveddownwardly by its support rod 15 for immersion in the well of the supplyplate while the pin 12 remains stationary at a higher elevation, FIG.9E, or it may assist in the resupply action, see FIG. 9L, describedbelow. The ring is moved by associated driver D₁, FIG. 9A.

At cleaning and drying stations, FIGS. 9F and 9G, the lower surfaces ofthe pin 12 and ring 14′ are shown vertically aligned (the ring hereshown as a cylindrical ring).

At washing station, FIG. 9F, the ring and pin may both be subjected toreciprocation in the vat of cleaning solution in the same or oppositevertical directions to assist the cleaning process. The wash station maybe an ultrasonic bath.

The multipurpose station illustrated in FIG. 9G is sized to receivedeposit pin 12 and supply ring 14. It has an annular nozzle 200 directedinwardly against the pin and ring to subject the parts to a conical flowfrom fluid sources such as compressed air, pressurized liquid andaerosols. The flow is directed past the parts 12, 14 to a trap havingdisposable filter 202 that intercepts material being removed from theparts. The trap may be associated with a vacuum pump. As shown, nozzle200 is associated with a secondary air path 204 to enable nozzle flow toinduce a flow of secondary air when desired.

The system of FIG. 9G is useful to remove sample liquid from the parts,to effect cleaning, and to dry the parts. For example the pin and ringare first exposed to one or more simultaneous or successive fluidcurrents or blasts of continuous or pulsed flow that blow remainingsample fluid from the parts and into the trap. Subsequently, a fluidstream of liquid or air may expose the parts to cleaning fluids such asliquid streams or aerosols containing water-borne detergent. This isfollowed by rinsing with pure water from the nozzle. Following washing,an air current from the nozzle, supplemented by induced air flow 204,can dry both pin and ring, in which case the air streams may be heated.

Supply from wells of 96 well plates, for deposit of the restrictedamounts of fluid that result from PCR (polymerase chain reaction)present a particular problem. Referring to FIG. 9H, wells 100 are madeto hold extremely small volumes of fluid, typically 2 to 10 micro liter(1 micro liter=1 cubic mm). These wells are typically cone-shaped withthe top diameter about 6 mm and the bottom shaped as a semisphere about2 mm in diameter. Liquids, even of low viscosity, for instance water,are so held by surface tension in such a well that volumes up to 15micro liter can be held against gravity when the plate is inverted.Smaller amounts of such liquids to supply a sub-reservoir ring aredifficult to extract from the narrow wells due to the aggregate effectsof surface tension, gravity, inertia and vacuum.

For removing liquid from such wells a supply ring 14 is provided with aspecial fluid retentive surface. One example is the provision ofinternal surface roughness of at least 1000 micro inch. This causes thecentral region of the ring effectively to have superior hydrophilicproperties, i.e. a better “grip” on the fluid by surface tensioneffects. This permits the uplift of a suitable volume of fluid from acontainer of approximately mating shape. The exterior surface of thering may also be provided with a fluid retentive surface to supplementsurface-tension effects of the ring, to compete with the retentiveproperties of the well.

Surface roughness of the internal or exterior surface can be obtained bysanding, broaching or by machining the part on a lathe with a tool or atap. The ring can also be manufactured from suitably coarse particulatematerial that is sintered or molded with a binder. Likewise a durablecoating can be applied such as formed by carbide particles.

As shown in FIG. 9I, in one preferred embodiment, 20 a cylindrical ring14A of stainless steel has a height h of 0.050 inch, an inner diameterof 0.060 inches and an outer diameter D of 0.080 inch. It is tapped by atool having 80 threads per inch, that produces a thread height d andpitch p of 0.012 inch, (the internal diameter is much larger than thediameter of the deposit pin P with which it is used). As shown in FIG.9H, an annular ring provided with surface roughness in this way iseffective to pick up liquid from the conical well of a PCR plate.Despite the desired surface tension effects produced by the internalring surface, it has smooth surface increments that promote goodcleaning.

Also shown in FIGS. 9I and 9J is support rod 15 e.g., of 0.15 inchdiameter stainless steel wire, soldered or spot welded at 104 a to theexterior of the ring. It drives the ring in its motions, and provideslateral and axial compliance for adapting to any misalignment entering anarrow well.

In FIG. 9K is shown a form of pin and ring assembly in which fluidcontacting surfaces of both ring 14 and pin 12 are defined by a specialsubstance 21 having a surface energy in excess of 2500 millinewton permeter mN/m), preferably provided by tungsten layers T.

FIGS. 9K and 9L show an advantageous relationship of pin and ring forresupply of the ring. When the ring is immersed in selected well W of amultiwell plate, the pin is present within the confines of the ring, tohelp the ring pick up the fluid. Their surface tension propertieseffectively cooperate to compete with the surface tension effects of thewalls of the well that resist removal of small quantities of the fluid.In the presently preferred relationship, the bottom tip surface of thepin is substantially aligned with the lower surface of the ring.Withdrawal of the assembly from immersion in well W withdraws a desiredamount of fluid, pendent as a large meniscal drop, bounded by the pickup ring, FIG. 9M. This quantity, protected and supported by the ring, isthen available for deposit in tiny drops by repeated projection of thepin through the ring, see dotted lines, FIG. 9K.

The sub-reservoir ring may have various advantageous forms such asaxially adjacent circular rings, multi-turn helical shapes, closedcylinders, open rectangular rings, open “U” shaped structures, etc. Thusthe term “ring” or “annular ring” as used generally refers to any closedor partially closed structure that, through surface tension effectsbetween adjacent or opposed surfaces, supports a volume of liquid in aspace through which a deposit device such as deposit pin 12 can operate.The size of the opening or bore of the ring, as well as the size, forinstance, of wire or ribbon that forms the cross-sectional shape of thering is selected in relation to the properties of the fluid (e.g.viscosity and surface tension), the number of deposits to be made from agiven fluid charge in the reservoir ring, and the size of the depositpin that is to move through the ring.

The size and shape of the deposit pins that cooperate with these andother sub-reservoirs also vary depending upon the application. It ispossible to employ pins of various transverse cross-section, e.g. squareor hexagonal or even rectangular or oval cross-section of equivalentarea to round cross-section pins. Especially for small dots, the pinsmay advantageously have stepped transverse cross-sections, e.g. may havean extremely small cross-section at the deposit end, to size thedeposited drop, stepped to a larger cross-section in the main body, forproviding structural stability. An example is shown in FIG. 2B.

For implementing the broad concept of a local, mobile supply, othertechniques than those shown can be employed. An example is a large diprod, an enlarged version of a deposit pin, from which a large dropdepends, which travels with the pin and is visited by the pin by asuitable motion, such as rotation.

C. Operating Systems

Some advantageous, novel operating systems that implement the foregoingprinciples will now be described.

Dip & Dot System

The mobile reservoir MW shown in FIG. 1 is shown multi-celled, torepresent a multi-well plate. Under computer control, an appropriate X,Ystage brings the chosen fluid resupply well in alignment under the pin.The pin is then controlled to descend, make contact with (dip into) thereservoir fluid and rise, taking a small amount of fluid in the form ofa pendant drop.

The pin is raised sufficiently to permit the pin and reservoir toseparate e.g. by computer controlled sideways movement of the reservoir,freeing the pin to descend unobstructed to deposit its small fluid dropon the targeted location on the substrate.

With appropriate transport motions of pin and multi-well supply, theprocess is repeatable at each location where a sample of the selectedfluid is desired, the fluid in the proper well being repeatedly broughtinto alignment with the proper pin for resupply and deposit in thedesired location by computer control. Each time a pin is commanded toreceive a fluid from a well different from that of its previous command,the pin is moved by computer control to a liquid removal, cleaning anddrying station, to prevent contamination.

For efficient operation, a multiplicity of pins may be used, see e.g.FIG. 24, at spacings that match the pattern of wells, enabling each pinto reach inside a separate well of the multiple well reservoir such as a96 well plate or a 384 well plate, as are known in the field ofbiochemistry and analytics.

The pin assembly and its driving mechanisms are preferably mounted on aprecision XY gantry as they require good positional accuracy. Themultiple well plate may be provided with two degrees of freedom in aplane parallel to the deposition plane and can be indexed under the pinassembly on a separate structure. Because of the relatively large sizeof the wells, the translation assembly for the plate may have lowerpositional accuracy than that of the pin. In the embodiment of FIGS. 11and 11A, however, the multi-pin assembly P_(A) and the mobile multiwellreservoir MW share the same X,Y gantry to advantage.

Rail support 60 of FIG. 11, constructed e.g. to support linear motormovement in the Y coordinate is mounted on an X stage 62, motor notshown. As shown, Y direction linear motors #1 and #2 respectively drivethe pin assembly P_(A) and the multiwell reservoir MW in the Ydirection. The reservoir has a secondary linear motor X₂ driven by afurther driver for X movement of the reservoir relative to the pinassembly. The pin assembly also has Z freedom of controlled movement,driven by a further driver Z.

Under computer control, the multiwell reservoir separates in the Ydirection from the pin assembly as shown in FIG. 11, and the Z stage isactuated to cause the pins to form deposits upon substrate R. Then, FIG.11A, the multiwell reservoir moves under the raised pins intoappropriate alignment, employing both Y₂ and X₂ motions under computercontrol. By Z motion the pins P_(A) dip into the commanded wells forresupply. The pins rise again, the multiwell reservoir moves laterallywith Y₂ motion out of the way and the deposit process is repeated at newtargeted X,Y location of the pins on substrate R or R₁. While thismobile reservoir technique is useful with pins of any construction, theadvantage of high accuracy of the linear motor indexing system isenjoyed when the pins are constrained in space to a highly accuraterepeatable position relative to their carrier, either with the highdensity pin arrangements made possible by the structures described withrespect to the various FIGS. 1, 2, and 3 or the flexure mountings thathave been described with respect to the FIGS. 4.

Multiple Pin Patterns

In the preferred embodiment of FIG. 12, two rows of 4 pins P, preferablyconstructed according to FIGS. 1-4 are spaced apart in a 9 mm squaregrid pattern matching the spacing of the wells of a 96 well plate. Thispermits transport of fluid from all 96 wells, 8 wells at a time to anassembled array of microscope slides, according to the scheme of FIGS.13 and 14, and directs the composition of 8 spaced apart blocks ofapproximate dimension each 8×8 mm on each slide, covering in totalapproximately 18×36 mm sq. Each pin deposits in a respective one of the8 blocks simultaneously with a single actuation of the Z drive. The headrepeats the action on each of the set of slides with the same fluid, andis then cleaned to be ready for fluid from different wells. The same pinmay be used to deposit the same fluid at a number of directed positionsin a given block, and/or upon the corresponding block of a number ofslides each having the set of 8 spaced apart blocks, the deposits on theslides being much closer than the spacing between wells. By followingthe sequence shown in FIG. 14, all wells may be visited by respectivepins. FIG. 15 and the magnified view of FIG. 15B show the array producedby the system and method described on a single slide. (Actually the dotsize in practice is much smaller than illustrated and dot density muchgreater, e.g., with as many as 50,000 or 100,000 dots carried by asingle slide.)

In a similar preferred embodiment, shown in FIGS. 16, 17, 18, 19, and19B a grid of 12 pins has 2 rows of 6 pins each, FIG. 16, again spacedapart in a 9 mm square grid pattern to match the spacing of the wells ofa 96 well plate. This arrangement permits the transport of fluid fromall 96 wells, 12 wells at a time, and directs the composition of 12spaced apart blocks of approximate total area 18×54 mm sq. FIG. 19 showssuch an array.

With either arrangement, the method is performed under computer controlto form a much more densely packed array of fluid dots than thatoccurring in the multiwell plates, e.g. arrays of 20 micron to 375micron diameter dots with similar spacing between dots, using all fluidsin the plate.

Just as the pins are located on 9 mm centers, the square arraysthemselves are distributed on 9 mm centers over the face of thesubstrate. By following the pickup sequence shown in FIG. 14 (rows 1through 2, and columns A through H), by repeated samplings, all wellsare visited, the pins being conveyed under computer control to thecleaning station, not shown, between change of fluids. The contents ofthe multiwell plate or a number of plates are thus distributed from thelow density distribution of wells in multiwell plates to high densityarrays.

Similarly, referring to FIGS. 16-19, again using 9 mm pin spacing, withtwo rows of 6 pins each, a sequence of samplings from the wells undercomputer control collects samples from all wells and uniquelydistributes them as high density array deposits in 12 squares on themicroscope slides or other substrate with array and slide dimensions asshown in FIG. 19.

The benefit of such groups of pins is to create a large number ofdeposited dots simultaneously on one or many microscope slides orsubstrates. This can substantially reduce the time and cost required tocreate high density arrays.

The assemblage of pins on a 9 mm square grid can also be used totransport fluid from plates with well spacing constructed on a squaregrid that is based on sub multiples of 9 mm, such as plates with 384wells or 864 wells or 1536 wells, etc. The high accuracy of the computercontrolled gantry system enables accurate placement of the selectedwells with respect to the pins, and the pins with respect to thereceiving substrate.

It is evident that using the same logic, pins can be assembled in denserconstructions to fit plates with smaller well spacings.

The denser the array, the tighter the location tolerances for thelocation of each small dot. The systems of laterally constrained depositpins described in FIGS. 1-4 are particularly capable of repetitiveproduction of precise high density arrays. Using these principles, themode of supplying the tips with fluid can be selected in reference tothe nature of the fluid as well as other operating parameters. A ringsupply mode will now be described.

Pin & Ring System

The pin assemblies as shown in FIGS. 1-4 can be used with a simple axialring translation mechanism. As the fluid needs to be picked up from arather large well, a sufficiently compact arrangement of multiple pinsand supply rings is possible. FIG. 20 shows the relationship of a pinand ring without their support or actuation mechanisms. Seen in FIG. 20are supply ring 14, pin tip 12 d, ring body 35 from which a support rodsegment 15 extends to the ring 14, pin shaft 12 b, the pin seat 13formed on pin body 12 a and pin guide 12 g. FIG. 20A shows a set of foursuch pin and ring assemblies. It is evident that any number can beassembled in this fashion. FIG. 21 depicts a 4 pin and ring assemblywhere one can see the pin holding structure, according to FIG. 2, andthe ring holding structure and their respective linear stepper motorsZ₁, and Z₂ that enable relative vertical motion. The Z₁ motion fordeposit on a receiving surface preferably involves overtravel, thecompliance of the deposit pins relative to the receiving surfaceensuring proper deposition over a range of surface heights. Therespective supporting linear guide rails for X and Y motion provide acomplete array-forming mechanism. FIG. 22 illustrates a commercialrealization of the design which attaches to the Y stage linear motor ofFIG. 21.

Referring to FIGS. 23, 23A, and 23B, deposit pin 12 in this case ismounted on a parallelogram, cantilever construction. Spaced-apart planarflexures 60 are mounted in parallel on a mounting block 62, sandwichedby mounting plates 64 and 67 against the intervening block 62. Theseflexures extend in cantilever fashion to intermediate block 66, arrangedto be engaged by pusher rod 68 associated with a prime mover 76, FIG.23B. Extending further in cantilever fashion from intermediate block 66are parallel flexures 70 and 72 which include cut-outs 74 that renderthe flexures weak and highly flexible (compliant). At the end 79 of weakflexures 70 and 72 is mounted deposit pin 12. The condition of no forcebeing applied to the structure is shown in FIG. 23 in which the flexuresare horizontal, the weight of the pin being borne by the mountingstructure. In FIG. 23A, force applied in the direction of the arrow 68results in deflection of the stiff flexures 60 to the shape shown, suchthat block 66 remains parallel to the receiving surface and deposit pin12 remains in perpendicular position to the receiving surface. (Thus therelatively stiff flexures 60 and the associated driver perform thefunction of a precision stage.)

The flexures may be comprised of synthetic resin cut to shape, e.g.polyamide resin, available as Kevlar™, from dupont, or etched from thinspring metal such as beryllium copper or stainless steel. Advantageouslyboth the stiff and weak flexures are formed continuously from a singlesheet of spring stock.

In FIG. 23B pusher 68 is driven by rotary motor 76 via lead screw, notshown. The sub-reservoir ring 14, mounted on support rod 15, likewise isdriven by motor 82 via a lead screw, for vertical motion of the ring.

The motor may advance the pusher 68 a. predetermined distance from ahome position for each deposition action, or to the level of a positionsensor which terminates the motion. The microscope slide or othersubstrate surface R may lie at slightly different levels due e.g. topermitted manufacturing tolerances. The stop-position of pusher 68involves sufficient overtravel to ensure contact of the deposit pin 12with a microscope slide or other object of the least thickness withinthe tolerated range of thicknesses. The compliance provided by weakflexures 70 or 72 (or the other arrangements discussed above), ensure,if the microscope slide or other substrate is considerably thicker thannormal, that the deposit force will still not exceed a predeterminedvalue, typically less than 1 gram, preferably less than 0.5 gram, forensuring precise dot formation and protection of the tip of the pin.

FIG. 24 shows a deposit cluster 28 of independently operated depositpins, formed by a number of the deposit assemblies described in theFIGS. 1-3 and 4. Cluster 28 includes, not shown, a number of independentdrives D and D′, one to drive each pin and one to drive each ring in Zdirection for picking up and depositing fluid, and sensors to indicateto the control electronics the position of the operative elements. Thereis a home sensor for each deposit pin 12 and a home sensor for each ring14. The devices are ganged mechanically for X or X,Y movement,positioned by a common electronic control. (Motion only in the Xdirection is employed when a stage is provided to advance the receivingsubstrate in the Y direction).

The cluster 28 may step to a selected X or selected X,Y position, atwhich a number of different motions under computer control may be causedto occur, picking up and depositing fluid in any order at any locationdesired. Such a cluster constitutes a particularly versatile tool whenemployed with conventional microtitre plates.

In such embodiments the aliquot carrier rings 14 and pins 12 are spacedin the cluster at 9 mm center-to-center distances or multiples thereofto facilitate operation with 96 well plates (in which the wells arespaced at 9 mm on center intervals, with 8 rows of 12 holes). Higherdensity plates also employ this configuration and have the samefootprint but employ more holes, 16×24, with hole-to-hole resistance of9/2 mm, to provide “384 plates”. The arrangement of FIG. 24 enables useof the higher density plates with existing automated 96 well platehandling equipment. The system described can be employed with both typesof plates, as well as any arbitrary arrangement.

The versatility of the cluster of independently operable deposit pins isillustrated by the following examples.

Sub-reservoir rings, e.g. set at 9 mm center-to-spacing, may be indexedin X,Y direction along with their pins and the rings may be driven down(or dropped) simultaneously for supply or resupply from four wells of aconventional 96 or 384 well plate, in an action similar to the systemspreviously described.

After suitable indexing, the four pins may be driven down simultaneouslyto form deposits at four places, in the same format as the supply plate.

Alternatively, during resupply, one sub-reservoir ring may be dropped topick up material from a selected well while all others remain in theirpassive positions. Then the cluster may be moved until the next ringarrives at the same well or another selected well, at which point it isdropped to pick up its aliquot, and so on, so that all of the rings mayhave the same fluid from the same wells or different fluid from anyselected wells.

The cluster 28 may be moved in X,Y direction between pickup or depositactions of successive pins so that, e.g. all of the pins deposit thesame or different fluid on a single slide at selectable addresses oreach pin addresses a different slide, but at a different location, ortwo pins address one slide and two another slide, or the deposits aremade one on top of another, etc.

The operator may also choose not to have one or more of the devicesoperating.

Thus it is seen that dense clustering of independently operable depositpins and rings according to the system of the FIGS. 1-3 and the systemsof the FIGS. 4 can enable high speed, versatile operation.

Actuation of all aliquot carriers simultaneously by one actuator and allpins actuated by another single actuator, to provide a multiple pinhead, realized with flexure-mounted pins, are shown in FIGS. 24A, 24Band 24D. Using linear stage techniques, two rows of four pins 12 at 9 mmspacing in both X and Y directions are all mounted on a frame 120 whichis reciprocated along rail 160 via carriage 162 by a single motor D.This causes the eight pins to move simultaneously. Likewise, two rows offour cooperating rings 14 are mounted on ring support 124, with the samespacing. The single support 124 is driven via carriage 126 by one motorD₁. In the embodiment shown, both embodiments share the same guide rail160. The pattern of dots shown in FIG. 24C is formed by a singleactuation of motor D, FIGS. 24A and D.

Arrayer

The gantry of an arrayer, now to be described, can carry one deposithead, a cluster of independently operable single pin heads, or amultiple pin head of the various designs described above. Combinationsof these are also possible.

FIG. 25 is a perspective view of a slide preparation machine forpreparing microscope slides or other substrates such as delicate soft orporous membranes carried on rigid supports. Its function is to rapidlydeposit a high density array of fluid dots of different compositions ona number of identical substrates, employing the microdot technology ofthe present invention. As shown in FIG. 25, there are four 96 wellsupply plates 31, serving as the central fluid source for resupply ofmobile fluid storage devices.

Horizontal base plate 200 provides a support structure to hold theoperating components. Fastened to base plate 200 are vertical sub plates210, 220, 230 and 240. Fastened to these plates is a dual axis motionsystem 250, comprising X and Y axis devices 260, 270 for providing X andY motions, in a parallel plane.

The guide rails of the X and Y axis devices, 260, 270 are parallel tobase plate 200, to carry deposit cluster 28 in X,Y motions in a planeparallel to base plate 200.

The X axis device 260 is a commercial device available from Adept ofJapan. It moves at a high rate of speed in a controlled manner using arotary servo motor with a drive screw and a shaft position encoder,employing digital and analog technology. Carried by X-axis device 260 isan orthogonally arrayed Y-axis device 270 which is a smaller versionthat operates in the same manner as the X-axis device.

The deposit cluster 28 comprises four deposition mechanisms, gangedtogether on a mounting structure as shown in FIG. 24. These devices maybe in accordance with the various structures shown.

After a deposition sequence is complete, the X and Y terminal drives thecluster of depositing elements to a cleaning station. In someembodiments they may be passed over the wells from which the fluidoriginated or other receptacle and subjected to air blast to dislodgeexcess fluid, or excess fluid may be removed by abrupt stopping of rapiddownward movement to dislodge excess fluid.

In the system of FIG. 25, the array of pins and rings of a cluster 28may be held over a vessel of water for cleaning, as shown in FIG. 9F.The vessel has water level and a pump constantly replenishes the water.Blotting paper or a cellulose sponge may be provided against which thepin and ring are blotted for fluid removal or drying.

Alternatively a fluid removal station according to FIG. 9G is employedwhere air flow removes remaining fluid from the pins and rings. Thearray of 4 pins thus purged of remaining fluid by air blast is then, bywarmed air, washed and rinsed by liquid or aerosol streams and dried.

FIG. 26 shows the control system of the machine. It shows the controlsfor the X and Y axis movement and also home center for the X and Y axis.The actual position of the carriage that the lead screw is driving issensed so the carriage can be driven home and then the counter isinitialized so precision motions can be made along both the X and Yaxes. Also shown is a schematic of the deposition head, one of many. Aspreviously described, each deposition head has two motors, a pin drivemotor and a ring motor, that are commanded from the control computer.

For deposit on microscope slides including slide-like rigid memberscarrying delicate, soft membranes, the slides are fastened to the table,or placed in register with guides in a known position. Features on thebase plate of the machine locate the slides in predeterminedorientation.

In the preferred embodiment of FIG. 25A, microscope slide MS rests uponslide support 30, having one end engaged with stop 31 and its other endengaged by a spring wire 32. Wire 32 extends from a support screw on thebottom side of support 30, through a hole 33 in support 30, and isbiased to the right in the figure to engage the slide MS to urge itagainst stop 31. The spring pressure is sufficient to hold the slide MSendwise in secure, accurate position despite vibrations that occurduring operation of the machine.

The slides are mounted side-by-side in subgroups of seven slides, withtheir thin long edges engaged with one another. The seventh slide'sposition is dependent only upon the tolerances of the preceding sixslides. By having such sub groups, one is assured that the array isproperly located. The computer is enabled to “talk” to the slide and torecord information, as in bar code. The bar code reader is mounted onthe servo drive 270 of the Y axis and adjacent to the deposition means28. The sequence starts with filling the multiplicity of rings of thedeposition device, and is carried out according to the control procedureof FIG. 27.

For use in high volume production contexts, the system described in theforegoing FIGS. 1-27 preferably employs a rapidly moving, laterallyconstrained, axially compliant pin, in a deposit cycle of less than 0.1second, in which impact and vibration is minimized, with the naturalfrequency of the system more than 10 Hz, in many cases preferably 20 Hz,a pin contact pressure of less than 1.0 gram, preferably less than 0.5gram in many cases preferably about 0.3 gram, and the system employingdamping.

Pin pressure on the substrate is light, and fluid splatter or separationconditions are thus avoided, despite the high speed of action, so thatdots of fluid of uniform shape are consistently formed at preciselycontrolled positions, even on soft or fragile receiving surfaces.

In the deposit action of the deposit pin, by raising the pin aftercontact of the drop on the substrate, the combined effects of inertia ofthe stationary fluid and surface tension (and of gravity, whendepositing downwardly, which is normally preferred) act upon the drop offluid to overcome the force of surface tension exerted by the liftingpin. The fluid drop preferentially stays with the surface of thesubstrate, and the pin, substantially devoid of fluid, is free to bereplenished and move rapidly to its next destination.

As the volume of the fluid is accurately specified by use of standardsizes of pin, and standard conditions, and the position of the pin isprecisely constrained, spots, dots and microdots of consistent size andprecise location are produced, that enable an improved degree ofquantification of observed results.

D. Examples of Novel Methods of Use

The systems described are useful with any native fragment of DNA, orpre-synthesized oligonucleotide of any length. There being norestriction as to chemicals, any non-photoreactive chemical as well asphotoreactive chemicals can be employed. Likewise dyes that are usefulto detect presence or absence of DNA may be selectively deposited inregistry with previously deposited spots or microdots of biologicalmaterial, and vice versa.

Among the many biological materials that may be spotted at high speedare fragments of nucleic acids, e.g. DNA, RNA or hybrids such as PNA(peptide nucleic acid), PCR (polymerase chain reaction) products, clonedDNA, and isolated genomic RNA or DNA, as well as synthetic analogs.

Also included are restriction enzyme fragments, full or partial lengthcDNA, mRNA or similar variations thereof, proteins such as proteinreceptors, enzymes, antibodies, peptides and protein digests;carbohydrates; pharmaceuticals; microbes including bacteria, virus,yeast, fungi, and PPLO; cells and tissue fragments; lipids,lipoproteins, and the like; plastic resin polymers, small particulatesolids in suspension, etc.

The deposition system may also be employed to deposit catalysts,reagents and encapsulents upon previously deposited material of any ofthe types above or, as mentioned below, to create an array of sites ormicro-wells for later reaction or growth of such material, or to assistin neutralizing or cleaning the deposit or reaction sites, as in thecase of highly toxic or virulent substances.

The most basic use of the arrayer is to create high density arrays ofnucleic acid on a porous or solid, flat surface, generally a microscopeslide or slide-like support. Deposit on fragile or soft surfaces such asmicroporous membranes or gels, glass cover slips, plastic surfaces, andwells of a microplate, or any substrate, which may be previously coatedor derivatized, may serve as a recipient surface.

In particular, membranes and gels are desirable to enable high densityanalysis with automatic equipment, using materials familiar to thefield, on which much of the important, historical data has previouslybeen acquired. Also, deposit on fragile glass cover slips is desirableas they are thinner than microscope slides, easier to maneuver, and whena beam of light is transmitted through them for transmission microscopy,better light capture occurs because the slip is thinner and less lightabsorptive. The system has the capability of spotting on plasticsurfaces without scarring or deforming the surface.

The avoidance of such surface deformation can be important, enabled byuse to low contact forces of the compliant pins. An undeformed surfacecan facilitate viewing with a confocal microscope, as it assures thatthe deposit remains in the plane of focus.

Use in wells of microplates is important. As has been mentioned, thenarrow lateral dimensions of the deposit pin, and its long length,enable deposit in multiple locations on the bottom of a well, or otherfluid containment region. For example the arrayer may be employed todeposit a number of spots in known locations on the bottom of a well toperform clinical tests on an analyte fluid. For instance, each spot inan array in the bottom of a well can be a known nucleotide probe. Asample added to the well will hybridize with spots with which the samplematches. For instance a diagnostic test may employ a 96 well plate tomeasure binding to as many as a hundred different probes printed inknown locations in the bottom of each well. Different patients' samplesmay be placed in respective wells, to conduct many evaluations at onceusing a single multiwell plate.

Another use of the system is to deposit, at useful speeds, astatistically determined number of molecules or units into a singlewell. Employing a suspension of suitable concentration in a supply ringwith an appropriately sized pin, thrusting the pin down once per well,statistically, can deposit the desired quantity, which then can interactwith nutrient, experimental drug, etc. in the well.

|The concept of insertion is extended to include the deposit ofparticulates in suspension, for example, to deposit cells and thenafterwards, deposit a suspension of particles of asbestos orprecipitated silica or other solids of interest, to investigate effectsof the particles upon the cells. These are examples of inexpensive,highly accurate, micro-controlled experiments that can be conducted atefficient speeds using the dedicated aliquot reservoir and deposit pin.

In many important cases the fluid or liquid carrier of the depositedspot evaporates and the biological or other material carried in thefluid stays in place by adhesive or bonding properties of the driedmaterial. In other cases, the spotting technique is useful to depositfluid that remains in a fluid state, for instance, as mentioned, todeposit cells into wells with fluid nutrient medium that enables thecells to continue to live.

In many cases it is important to know where a deposit is and that itwill stay in the deposited position when covered by a common reagent.Steps can be taken to secure the deposit in position, for instance, withDNA, by exposing the deposit to UV radiation to crosslink the materialor to use a derivatized surface that produces crosslinking between e.g.DNA and the surface on which it is deposited. An example is a silenatedsurface coated with E.S. aminosilene, to provide a positively chargedsurface which binds, by ionic or electrostatic forces, with negativelycharged deposits such as DNA.

In addition to applicability in bioresearch and clinical diagnosis, thedeposition system has applicability in the chemical laboratory, e.g. toanalyze fluids, such as for water quality, or to experiment with resins,for instance polymerization reactions , to conduct experiments in smallquantities of many different varieties, e.g. to determine optimum ratiosand optimum selection from a host of slightly varying examples. Therange of usefulness is broad with application to small quantities,different temporal sequences, different kinetics of reaction, anddifferent mixtures. In all of these cases, the system is a precise wayof manipulating small amounts of liquid, solids in liquid suspension andcells in suspension, under controlled conditions.

Deposition with the systems described leads to rapid and preciseobservations, reduction in the number of trials for a given experimentand improvement in the statistical significance of the data. Costsavings and improved experimental procedures can be realized.Quantification of results at accuracies heretofore unknown may beattained by consistent and precise dot formation that enables improvedsignal-to-noise ratio in detection, when sensing the difference between,e.g., the fluorescence of a deposited spot and the immediately adjacentbackground surface of the substrate.

The system is useful in many environments due to the attributes of thedeposit apparatus, and the techniques by which movement and control iseffected. The following are further examples.

The system is used to deposit dots of fluids of high volatility such asalcohol-based fluids, upon rigid substrates such as glass or silicon,upon membranes, etc. The relatively large mobile local reservoir ringthat travels with the deposit pin to the deposit site presents arelatively small exposed surface-to-mass ratio, which limitsevaporation. Transport from that volume of the tiny sample on the tip ofthe pin, over a short local distance, limits exposure of the tiny sampleto evaporating conditions until the dot of fluid is deposited.

Where desired, the operating deposit mechanism is shielded from windageby a protective shield mounted on the head to move in X-Y directionswith the deposit mechanism, to further limit evaporative loss. Inanother case, the environment in which the system operates iscontrolled, e.g. at high humidity, or high partial pressure of thevolatile substance, to limit evaporative loss, or at particularcontrolled conditions, e.g. controlled temperature and humidity, tofavor the deposition process or the operation of the system itself.

Time-based sampling to evaluate. chemical reactions or growth stages canbe performed automatically without attendance of laboratory personnel.In one example, the fluid carrier ring through which the pin operates isemployed as a reaction vessel from which samples of the continuingreaction are periodically taken by an associated deposit pin, anddeposited for later inspection.

In this or other examples, at prescribed time intervals, another pinmoves through its ring to deposit an inhibiting reagent to halt thereaction or growth that is occurring at a respective location on asubstrate. By doing this at timed intervals over different locations onan array of identical reactions, a fixed array that represents thesequence of conditions at the various time intervals is preserved forlater examination.

In another method employing the deposit system, an etchant fluid isprovided in a local reservoir ring. The pin of the deposit pindistributes the etchant in tiny, precise spots or microdots in a desiredarray across a reactive substrate surface. For instance, for formingmicro-wells for containing fluid, the device deposits an acid such ashydrochloric acid in an array of small dots upon a silicon substrate. Anetching reaction occurs, and the substrate is then neutralized andwashed, to produce a corresponding array of small wells. These may haveadvantageous hydrophilic, fluid-retaining surfaces as a result of theetching process. Following this, the same depositing system may beemployed to deposit one or more substances precisely in registrationwith each of the wells for use in reaction or growth processes that aredesired. Plates thus prepared may be transferred e.g. to a wide fieldscanning microscope for observation.

Arrayers as described can also be used for color printing of fabrics,paper etc., where the 96 well plate holds different color inks or dyes.The area to be printed is the entire reach of the gantry less the colorsource and washing station.

The arrayer can be used to generate a single printed circuit board,e.g., prototype boards, or boards for limited volume production, wherethe machine is employed to deposit varnish or photoresist or otherprotective coating material to define the regions of the copper clad orother substance which need to be preserved from acid etching. Likewisethe arrayer may be employed to deposit photoactive substance forproduction of “biological” deposits using lithographic techniques.

E. Combination Arraying and Microscopic Analysis

It is an important further feature of the invention to combine thearrayer of any of the presented embodiments, or its steps of action, orarray products of its operation, with a scanning microscope, especiallya wide field scanning microscope such as available from applicant. Theprinciples described here enable wide area arrays to be formed of veryhigh density over the mentioned wide range of fluids and conditions,while wide area scanning microscopes enable commensurate accurate andinexpensive reading of the results achieved with such wide arrays. Thewide area and precision capabilities of each system and method, incombination, complements the other to achieve an enabling, significantadvance in microdot reaction and analysis. Use of an array offlurophor-tagged components, such as is employed in biotechnology, orflurophor-tagged contaminants, followed by reading with a suitable widefield scanning microscope that excites the flurophors is of particularadvantage.

F. Useful Additional Features

In one embodiment, an inductive heater station is provided to which thedeposit mechanism can travel under computer control. In this case thesubstance of the reservoir ring and the deposit pin, or at least thesurface portions of these devices, are comprised of electricallyconductive material capable of having electrical currents induced by analternating field of the induction heater. Under computer control, thereservoir ring and the pin are delivered for a momentary pause in theheater, for heating based on resistive (I²R) losses by the inducedelectrical currents, for instance to sterilize the reservoir ring anddeposit pin or to stop bioactivity in the fluid material retained on theinstruments.

In another instance, a reservoir ring containing a charge of reactantfluid, which is desired to be heated, can be introduced to the inductiveheater, and the fluid is heated by heat-transfer to the fluid from theinductively heated ring. Such heating can be employed to initiate areaction in the fluid, for subsequent deposit.

Another system includes a delivery system for relatively largerquantities of fluid, e.g. to fill a micro-well with nutrient, diluent orreagent after deposit of a spot of the fluid of interest. The deliverysystem, such as a computer-controlled pipette, may be associated on thesame head and X-Y carriage with the deposit pin, or in a separate heador carriage. By functioning under computer control to deliver largerquantities of fluid to reaction sites where dots of fluid havepreviously been deposited, an entire experiment can be automated. Fluidswhich may be introduced in this way include, for instance, solvents,etchants, sterilizing agents, cleaning agents, encapsulating coatingmaterials, etc.

In another method the deposit pin is caused to deposit reagents atselected sites in differing amounts at differing locations, toeffectively conduct titration, to observe a reaction at differentconcentrations of the reagent. Thus, at one reaction site (e.g. a flatarea or a well on a substrate) the deposit pin may deposit one precisedrop of reagent, at a second see two precisely identical drops of thereagent, at a third selected site three precisely identical drops of thereagent, and so on, to provide the full range of concentrations desiredfor evaluating reaction of the reagent with another substance that hasbeen preapplied to the site or that is subsequently applied.

While such systems are particularly well suited for laboratoryexperiments, they also can be employed in industrial process control.

A variation of the spotter mechanism employs, in a fashion analogous tothat of a modern milling machine, a set of interchangeable heads havingdifferent capabilities. Under computer control, an X-Y carriage of thesystem is moved to select a desired head which is carried across thesubstrate to perform its function. In some instances the device selectedmay be a sub-reservoir ring from a set of such rings that have differentinternal diameter or are formed of different wire or ribbon sizes, orare of different sizes to enter different wells, etc. These provide avariety of carrying capacities for fluids of different viscosities orfor use with deposit pins of different sizes. Likewise, different sizesof deposit pins can be selected from a set of pins to vary the size ofthe spot to be deposited. Heads can also be selected that provide otherdevices for preparing for or conducting experiments or for theproduction of reference or diagnostic well plates and slides.

In some cases the selection and use of devices can be conducted undercomplete computer control to enable automatic performance of amulti-task experiment un-attended by the technician.

In addition to depositing spots of fluid upon a standard microscopeslide, and upon porous or soft membranes and other delicate substrates,it is possible and advantageous to deposit spots on substrates ofsignificantly larger area and on other substances and on surfaces havingspecial formations, for instance upon substrates having micro-cavitiesthat have been formed by the instrument itself, by one of the techniquesdescribed above. Plates delivered with the micro-cavities preformed inthe substrate may also be used, and aligned for deposit of fluid byautomatic controls of the instrument, or the control system of the unitis advantageously provided with a vision system that “reads” thelocation and pattern of the array of micro-wells, and adjusts itselfautomatically or under operator control to accurately deposit dots offluid in wells.

F. Conclusion

In the various ways described, a large array of fluid deposit sites maybe established and managed in a precise, repeatable manner that employsthe same concentrations or reactions or precisely varied concentrationsand reactions. This may be done to enable examination, to promotereaction or growth processes in biotechnology, life sciences, chemistry,pollution detection, process control and in industry in general.

Thus, beyond an instrument for low-cost preparation of microscope slidesand membranes for biotechnology research, there has been contributed auniversal and widely variable set of systems, instruments, methods andproducts that can advance research and industry.

Numerous other embodiments not described in detail here can apply theprinciples described to particular applications and are within the scopeof the claims.

1-26. (canceled)
 27. An apparatus for depositing fluid dots on areceiving surface in an array, comprising: a deposit devicecooperatively related with a fluid source; a multiplicity ofdrop-carrying elements coupled to the deposit device; a transportmechanism for positioning the device at a precisely referenced positionover the receiving surface; and a drive mechanism for moving theelement, relatively, in deposition motion toward and away from thesurface.
 28. The apparatus of claim 27 further including a multiplicityof fluid-retaining structures, each co-operatively arranged with thedrop-carrying element and constructed to retain fluid includingbiological material by surface tension.
 29. The apparatus of claim 28,wherein each said fluid-retaining structure is constructed and shapedfor at least partial immersion into a well including the biologicalmaterial.
 30. The apparatus of claim 29, wherein said fluid-retainingstructure includes a circular-shaped member for said retention of liquidby surface tension and said drop-carrying element is constructed to movewith respect to said member to receive said fluid therefrom.
 31. Theapparatus of claim 29, wherein said fluid-retaining structure includes aU-shaped member for said retention of liquid by surface tension and saiddrop-carrying element is constructed to move with respect to said memberto receive said fluid therefrom.
 32. The apparatus of claim 29, whereinfluid-retaining structure includes a helical member for said retentionof liquid by surface tension and said drop-carrying element isconstructed to move with respect to said member to receive said fluidtherefrom.
 33. The apparatus of claim 29, wherein fluid-retainingstructure includes a closed shape member for said retention of liquid bysurface tension and said drop-carrying element is constructed to movewith respect to said member to receive said fluid therefrom.
 34. Theapparatus of claim 29, wherein fluid-retaining structure includes apartially closed shape member for said retention of liquid by surfacetension and said drop-carrying element is constructed to move withrespect to said member to receive said fluid therefrom.
 35. Theapparatus of claim 29, wherein fluid-retaining structure is made of amaterial providing appropriate surface tension for retaining said liquidincluding biological material and said drop-carrying element isconstructed to move with respect to said member to receive said fluidtherefrom.
 36. The apparatus of claim 29, wherein said fluid-retainingstructure includes a circular-shaped member for said retention of liquidby surface tension and said drop-carrying element is constructed to movewith respect to said member to receive said fluid therefrom.
 37. Theapparatus of claim 29, wherein said drop-carrying element includes apin.
 38. The apparatus of claim 29, wherein said deposit device isconstructed to urge said drop-carrying element to a predeterminedposition to achieve said precisely referenced position when saiddrop-carrying elements is in contact with the receiving surface.
 39. Theapparatus of claim 38, wherein said deposit device is constructed toachieve said precisely referenced position using a gravity element. 40.The apparatus of claim 38, wherein said deposit device is constructed toachieve said precisely referenced position using a spring.
 41. Theapparatus of claim 27, wherein the receiving surface includes a rigid,smooth substrate.
 42. The apparatus of claim 41, wherein the rigid,smooth substrate is a glass slide.
 43. The apparatus of claim 27,wherein the receiving surface includes a porous membrane.
 44. Theapparatus of claim 27, wherein the receiving surface includes anitrocellulose.
 45. The apparatus of claim 44, wherein the receivingsurface includes a cellulose acetate, polyvinylidine fluoride (PVDF) ornylon.
 46. The apparatus of claim 45, wherein the receiving surfaceincludes a gel.